Recombinant Marchantia polymorpha Photosystem II reaction center protein H (psbH)

What is Marchantia polymorpha and why has it emerged as a valuable model organism for chloroplast protein research?

Marchantia polymorpha is a complex thalloid liverwort and an extant relative of the earliest terrestrial plants. It has gained significant attention as a model organism for several compelling reasons. First, M. polymorpha possesses a relatively simple genome with remarkably low genetic redundancy compared to higher plants like Arabidopsis thaliana, making it easier to study gene function without the complication of redundant gene families . Second, it has a dominant haploid gametophytic lifestyle, simplifying genetic analyses as recessive mutations are directly expressed in the haploid tissue .

The organism is dioecious (having separate male and female plants) and reproduces both vegetatively through propagules and sexually through spores that can develop from single cells in culture . For chloroplast protein studies specifically, M. polymorpha offers unique advantages including the absence of RNA editing mechanisms in its chloroplasts, providing a convenient test-bed for studying genetic elements involved in plastid gene expression . Additionally, an extensive array of molecular techniques has been developed for M. polymorpha, including efficient transformation methods, vectors, markers, homologous recombination, and CRISPR-Cas9 mutagenesis approaches .

What is the psbH gene and what role does it play in photosynthetic function?

The psbH gene encodes a small protein component of the Photosystem II (PSII) complex, a crucial multiprotein assembly responsible for light-driven water oxidation during photosynthesis. The psbH gene in chloroplasts is part of the polycistronic psbB-psbT-psbH-petB-petD operon, which undergoes complex RNA processing events to produce mature oligocistronic RNAs .

This gene encodes a small transmembrane protein that appears to play an important role in the assembly, stability, and/or repair of the PSII complex. Though relatively small compared to other PSII components, psbH likely contributes to the structural integrity of the complex and may be involved in regulating electron transport processes. Mutations affecting psbH expression or processing have been shown to impact photosynthetic efficiency, highlighting its functional significance despite its modest size.

In the context of the PSII complex assembly, psbH is integrated in the later stages of biogenesis after the formation of the initial reaction center (RC) complex, which consists of D1, D2, PsbI, and cytochrome b559 subunits . The exact timing and mechanism of psbH incorporation into the growing PSII complex in M. polymorpha remain areas of active investigation.

How is the psbB-psbT-psbH-petB-petD operon organized and processed in chloroplasts?

The psbB-psbT-psbH-petB-petD operon represents a classic example of a complex chloroplast transcriptional unit that undergoes extensive post-transcriptional processing. This operon contains genes encoding key components of both the PSII complex (psbB, psbT, and psbH) and the cytochrome b6f complex (petB and petD).

The primary transcription of this operon produces a pentacistronic RNA that undergoes multiple processing events including RNA splicing (for intron-containing genes like petB) and intercistronic cleavage to generate various oligocistronic and monocistronic transcripts. A comprehensive analysis of the RNA species derived from this operon reveals:

• Dicistronic psbB-psbT RNAs of approximately 1900 and 2000 nucleotides (representing two different psbB RNA 5′ ends)

• Tricistronic psbH-petB-petD RNA of approximately 1800 nucleotides

• Dicistronic petB-petD transcript of approximately 1500 nucleotides

• Monocistronic petB RNA of approximately 800 nucleotides

• Monocistronic psbH RNA of approximately 400 nucleotides

Studies with the Arabidopsis hcf152 mutants have revealed that specific nuclear-encoded factors are required for the processing and expression of these transcripts. For instance, the hcf152-1 mutation affects RNA processing between psbH and petB, leading to depletion of RNAs processed between these genes . This includes loss of the 2600-nucleotide psbB-psbT-psbH RNA, the 400-nucleotide monocistronic psbH RNA, and the dicistronic 2200-nucleotide petB(IB)-petD RNA .

What methodological approaches are most effective for recombinant expression of psbH in Marchantia polymorpha?

For successful recombinant expression of psbH in M. polymorpha, researchers should consider both nuclear and chloroplast transformation strategies, each with distinct advantages depending on research objectives:

Chloroplast Transformation Strategy:
For chloroplast expression, biolistic transformation has proven highly effective with M. polymorpha spores . The development of codon-optimized fluorescent reporter genes like mturq2cp has significantly improved the ability to identify and characterize transplastomic events . For optimal expression of psbH, the following promoter-UTR combinations have demonstrated high efficiency:

• The hybrid Nt-psbA promoter and 5'UTR with the Mp-rbcL sequence

• The native Mp-rbcL promoter-5′UTR sequence

Both these regulatory elements support high expression levels with minimal deleterious effects on growth, making them promising choices for psbH expression . When designing expression constructs, it's important to note that protein hyperexpression from the chloroplast genome can lead to reduced growth rates, with transformed lines expressing up to 15% of total soluble protein showing some growth penalties .

Nuclear Transformation Approach:
For nuclear transformation, Agrobacterium-mediated methods with selection markers like hygromycin or G418 resistance are recommended. When expressing psbH from the nuclear genome, consider the following promoters which show distinct expression patterns:

• Alpha tubulin-like promoter - preferentially expressed in meristematic areas

• RuBisCO small subunit-like promoter - mainly detected in photosynthetic tissues

• Ubiquitin-like promoter - expressed throughout the gemmae

Each promoter provides different spatial expression patterns that can be selected based on experimental requirements. For efficient localization of the expressed psbH to chloroplasts, inclusion of a chloroplast transit peptide is essential.

How can researchers effectively analyze psbH integration into functional PSII complexes?

Analyzing the successful integration of recombinant psbH into functional PSII complexes requires a multi-faceted approach combining biochemical, spectroscopic, and functional assays:

Biochemical Analysis:

• Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) followed by second-dimension SDS-PAGE represents a powerful technique for resolving intact PSII complexes and subcomplexes. The PSII reaction center-like complex containing D1, D2, PsbI, and cytochrome b559 migrates at approximately 150 kD in BN-PAGE . Researchers should look for co-migration of psbH with these core components.

• Immunoblot analysis using antibodies against psbH and other PSII components can confirm the presence of psbH in isolated PSII complexes. This approach is particularly useful when comparing wild-type and mutant or transgenic plants.

Spectroscopic Methods:

• Chlorophyll fluorescence measurements provide a non-invasive tool for assessing PSII function. Parameters such as Fv/Fm (maximum quantum yield of PSII) and ΦPSII (effective quantum yield) can indicate whether psbH incorporation has resulted in functional PSII complexes.

• Low-temperature (77K) fluorescence emission spectra can distinguish between properly assembled PSII complexes and free chlorophyll molecules or partially assembled complexes.

Functional Assays:

• Oxygen evolution measurements directly assess the water-splitting activity of PSII, providing quantitative data on complex functionality.

• Electron transport rate measurements using artificial electron acceptors can evaluate the electron flow through PSII complexes containing the recombinant psbH.

What role do auxiliary proteins play in facilitating psbH incorporation into the PSII complex?

The assembly of PSII is a highly coordinated process involving numerous auxiliary proteins that assist in the incorporation of individual components like psbH. Research has identified several key factors that play critical roles in this process:

OHP1 and OHP2 Proteins:
The ONE-HELIX PROTEIN1 (OHP1) and OHP2 are essential for the formation of the PSII reaction center (RC) . These proteins contain one light-harvesting chlorophyll a/b-binding (LHC) domain and appear to function in chlorophyll binding in vivo, as evidenced by mutagenesis studies of their chlorophyll-binding residues . In Arabidopsis, plants lacking either OHP1 or OHP2 show pale-green phenotypes and impaired accumulation of photosystems .

HCF244 Protein:
HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244) forms a functional complex with OHP1 and OHP2 . Biochemical analyses show that OHP1, OHP2, and HCF244, together with D1, D2, PsbI, and cytochrome b559, form a PSII RC-like complex distinct from the RC subcomplex in the intact PSII complex . This complex appears to function transiently at an early stage of PSII de novo assembly and during PSII repair under high-light conditions .

Temporal Dynamics of Auxiliary Proteins:
Immunoblot analysis of thylakoid membrane complexes separated by 2D BN/SDS-urea-PAGE reveals that OHP1, OHP2, and HCF244 co-migrate in a complex of approximately 150 kD, which also contains traces of D1, D2, PsbE, and PsbI . This suggests a sequential assembly process where these auxiliary proteins are present in the PSII RC-like complex for a limited time before being released and replaced by other PSII subunits, including psbH .

How does the absence of RNA editing mechanisms in Marchantia polymorpha impact psbH expression and function?

Marchantia polymorpha provides a unique experimental system for studying chloroplast gene expression due to the absence of RNA editing mechanisms, which are present in most land plants . This characteristic has several important implications for psbH expression and function:

Simplified Post-transcriptional Processing:
The absence of RNA editing eliminates one layer of post-transcriptional regulation, potentially making the expression of psbH more straightforward in M. polymorpha compared to other plant species. This feature makes M. polymorpha an attractive model for studying the basic mechanisms of psbH expression without the confounding effects of RNA editing.

Codon Usage Considerations:
Without RNA editing to modify certain codons post-transcriptionally, the genomic sequence of psbH in M. polymorpha directly determines the amino acid sequence of the protein. This characteristic necessitates careful consideration of codon optimization when designing recombinant psbH constructs, particularly when transferring sequences from species that utilize RNA editing.

Experimental Advantages:
The absence of RNA editing makes M. polymorpha an excellent experimental system for studying cis-regulatory elements involved in psbH expression. For example, researchers can directly assess the impact of modifications to promoter regions, untranslated regions (UTRs), or coding sequences without having to account for potential RNA editing events that might alter the expressed sequence.

Evolutionary Implications:
Comparative studies of psbH function between M. polymorpha and species that employ RNA editing can provide insights into the evolutionary significance of RNA editing for photosynthetic gene expression. Such studies might reveal whether RNA editing provides functional advantages for psbH expression or whether it represents an evolutionary constraint that M. polymorpha has circumvented.

What strategies can be employed to optimize the stability and accumulation of recombinant psbH protein?

Optimizing the stability and accumulation of recombinant psbH protein in M. polymorpha requires addressing several challenges related to protein expression, folding, and integration into multiprotein complexes:

Promoter and 5' UTR Selection:
The choice of promoter and 5' untranslated region significantly impacts expression levels. For chloroplast expression, both the hybrid Nt-psbA promoter with 5'UTR Mp-rbcL and the native Mp-rbcL promoter-5′UTR have demonstrated high activity with minimal deleterious effects on growth . When targeting nuclear expression with subsequent chloroplast import, the RuBisCO small subunit-like promoter shows preferential expression in photosynthetic tissues where psbH function is most relevant .

Co-expression with Assembly Factors:
Co-expressing psbH with its known assembly partners or chaperones can enhance its stability and proper incorporation into PSII complexes. Given the evidence that OHP1, OHP2, and HCF244 form a transient complex with PSII reaction center components , co-expression of these factors might facilitate proper psbH incorporation.

Protection from Proteolytic Degradation:
Unassembled or misfolded membrane proteins like psbH are often targeted for proteolytic degradation. Strategies to minimize degradation include:

• Expression under controlled light conditions to coordinate with the synthesis of other PSII components

• Co-expression with proteins that can bind to and stabilize psbH

• Modification of sequences that might serve as proteolytic recognition sites without altering functional domains

Conditional Expression Systems:
To mitigate potential growth penalties associated with protein hyperexpression in chloroplasts, conditional expression systems may be useful. These could function through:

• Regulation of transcription in the chloroplast

• Regulation of mRNA stability through conditional expression of heterologous PPR proteins

• Supplementary expression of potentially limiting PPR proteins