Recombinant Pisum sativum NAD (P)H-quinone oxidoreductase subunit 5, chloroplastic (ndhF)

What is the role of NAD(P)H-quinone oxidoreductase subunit 5 (ndhF) in Pisum sativum chloroplasts?

NAD(P)H-quinone oxidoreductase subunit 5 (ndhF) is a critical component of the chloroplast NAD(P)H dehydrogenase (NDH) complex in Pisum sativum. This complex mediates both photosystem I (PSI) cyclic electron transport and chlororespiratory electron transport pathways. The NDH complex helps prevent overreduction of stroma, particularly under stress conditions, by facilitating alternative electron flow pathways . As part of this complex, ndhF contributes to the membrane domain of the NDH complex and is encoded by the plastid genome (plastome). The protein functions as part of the machinery that oxidizes NADH and/or NADPH and reduces plastoquinone in the thylakoid membrane.

How does the pea NDH complex differ structurally from cyanobacterial NDH and mitochondrial Complex I?

The NDH complex in pea chloroplasts shares structural similarities with both cyanobacterial NDH and mitochondrial Complex I but has evolved unique characteristics. Research has revealed that the chloroplast NDH complex can be divided into four subcomplexes:

• Subcomplex A - Conserved in cyanobacteria

• Membrane subcomplex - Conserved in cyanobacteria

• Subcomplex B - Specific to chloroplasts

• Lumen subcomplex - Specific to chloroplasts

The evolution of novel subcomplexes B and lumen appears to be specific adaptations in land plants, possibly facilitating more efficient operation of NDH . Unlike the cyanobacterial counterpart, the pea NDH complex forms a supercomplex with PSI (NDH-PSI), which requires two minor light-harvesting complex I proteins, Lhca5 and Lhca6, for full assembly. The isolated Ndh complex from pea has an approximate molecular mass of 550 kDa and consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH .

What are the optimal methods for isolating active recombinant ndhF protein from Pisum sativum?

Isolation of active recombinant ndhF requires a carefully optimized protocol that preserves the native conformation and activity of the protein. Based on established methodologies for similar proteins in Pisum sativum, the following approach is recommended:

• Gene cloning and expression system selection:

  • Clone the ndhF gene from Pisum sativum

  • Express in E. coli as a fusion protein (similar to the approach used for NADPH:protochlorophyllide oxidoreductase from pea)

  • Consider fusion partners like maltose-binding protein (MBP) that can improve solubility

• Purification strategy:

  • Selective solubilization of thylakoid membranes with mild detergents like dodecyl maltoside

  • Sequential chromatography:

    • Anion-exchange chromatography (e.g., DEAE-cellulose)

    • Size-exclusion chromatography

    • Affinity chromatography using tagged constructs

• Activity preservation:

  • Maintain buffers containing stabilizing agents

  • Use glycerol in storage buffers (typically 50%)

  • Avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

When assessing protein purity and activity, researchers should employ a combination of SDS-PAGE, immunoblotting with anti-NdhF antibodies, and enzymatic activity assays using ferricyanide or quinones as electron acceptors.

What enzymatic assays can be used to verify the functional activity of purified ndhF in the context of the NDH complex?

Several enzymatic assays can be employed to verify the functional activity of ndhF as part of the NDH complex:

• NADH/NADPH dehydrogenase activity:

  • Measure NADH and/or NADPH oxidation spectrophotometrically at 340 nm

  • Test with various electron acceptors:

    • Ferricyanide

    • Menadione

    • Duroquinone

    • Natural plastoquinone

• Electron transport measurements:

  • Monitor reduction of artificial electron acceptors

  • Typical reaction conditions:

    • Buffer: Tris-based (pH 7.5-8.0)

    • NADH concentration: 50-200 μM

    • Electron acceptor (e.g., ferricyanide): 1 mM

    • Temperature: 25-30°C

• Diaphorase activity:

  • Measure reduction of 2,6-dichlorophenol indophenol (DCPIP)

  • Follow absorbance decrease at 600 nm

Typical experimental results for the NDH complex purified from pea show that it exhibits specific NADH- and deamino-NADH-dependent dehydrogenase activity characteristic of complex I. This activity can be observed when using either ferricyanide or quinones (menadione and duroquinone) as electron acceptors .

Note: Values are approximate and based on typical results from purified pea NDH complex.

How does the supercomplex formation between NDH and PSI impact the functional properties of ndhF?

The NDH-PSI supercomplex formation represents a sophisticated molecular adaptation that enhances the efficiency of cyclic electron transport. This formation impacts ndhF's functional properties in several ways:

• Altered electron transfer kinetics:

  • Direct channeling of electrons between complexes

  • Reduced diffusion limitations for electron carriers

  • Enhanced coupling of proton translocation to electron transport

• Structural rearrangements:

  • NDH component subunits, including ndhF, may undergo conformational changes when integrated into the supercomplex

  • These changes could modify substrate binding sites and catalytic properties

  • Interaction surfaces between NDH and PSI may stabilize certain conformations of ndhF

• Regulation mechanisms:

  • Supercomplex formation provides additional regulatory points for cyclic electron flow

  • Light-harvesting complex proteins Lhca5 and Lhca6 are required for full supercomplex assembly

  • Loss of these components results in formation of sub-supercomplexes with altered properties

Research using blue native (BN)-PAGE has identified the NDH-PSI supercomplex as a high molecular weight band (>1000 kDa). Mass spectrometry analysis of this complex has detected components from both NDH and PSI, confirming their physical association . Functional studies comparing wild-type plants with mutants lacking components needed for supercomplex formation (such as lhca6) show that supercomplex formation enhances NDH activity, particularly under stress conditions.

What role does ndhF play in the electron transfer pathway within the NDH complex?

The ndhF subunit (NAD(P)H-quinone oxidoreductase subunit 5) occupies a strategic position within the membrane domain of the NDH complex, contributing to the electron transfer pathway in the following ways:

The electron flow in the NDH complex follows a pathway from NAD(P)H through protein-bound cofactors (likely including iron-sulfur clusters) and ultimately to plastoquinone. While the exact electron transfer pathway in plant NDH complexes is not fully characterized, research on analogous complexes suggests that ndhF is positioned to participate in the later stages of this electron transfer chain, potentially contributing to plastoquinone binding and reduction.

How conserved is the ndhF gene across different plant species compared to Pisum sativum?

The ndhF gene shows an interesting pattern of conservation across plant lineages, with significant implications for evolutionary biology and functional studies:

• Conservation within land plants:

  • High sequence similarity among closely related legumes (70-95%)

  • Moderate conservation (50-70%) across broader angiosperm groups

  • Present in most land plants but with notable exceptions

• Selective loss in some lineages:

  • Completely absent in some parasitic plants

  • Lost in certain orchids and some gymnosperm lineages

  • Notably absent in Chlamydomonas reinhardtii

• Functional constraints on sequence evolution:

  • Transmembrane domains show higher conservation than loop regions

  • Quinone-binding regions display stronger sequence constraints

  • N-terminal and C-terminal regions show greater variability

Comparison of pea ndhF with other plant species reveals patterns of conservation reflecting both functional constraints and evolutionary history. The gene maintains higher similarity with other legumes but shows significant divergence from more distant plant groups, particularly in regions not directly involved in catalytic function.

What approaches should be used to investigate structure-function relationships in ndhF through site-directed mutagenesis?

Investigating structure-function relationships in ndhF through site-directed mutagenesis requires a systematic approach:

• Target selection strategy:

  • Focus on highly conserved residues first

  • Target predicted transmembrane domains and quinone-binding regions

  • Examine homology models based on bacterial and mitochondrial Complex I structures

  • Consider charge-altering substitutions in potential proton channels

• Expression system optimization:

  • Express recombinant protein as a fusion with maltose-binding protein (MBP) or similar partners to improve solubility

  • Use E. coli expression systems with optimized codons for plant proteins

  • Consider baculovirus expression for more complex structures

• Functional assays for mutants:

  • Develop a rapid screening assay for NDH activity

  • Compare NADH and NADPH oxidation rates

  • Measure electron transfer to various acceptors

  • Assess complex assembly through BN-PAGE

• Analysis of structural perturbations:

  • Use circular dichroism to assess secondary structure changes

  • Employ limited proteolysis to probe conformational changes

  • Analyze thermal stability of mutant proteins

A systematic mutagenesis approach should target residues in predicted functional domains, particularly those conserved across species. When designing experiments, researchers should consider that the ndhF protein functions as part of a multiprotein complex, so mutations might affect both intrinsic activity and interactions with partner proteins. The experimental design should include appropriate controls, including conservative mutations that preserve charge and size properties.

How does the activity of NDH complex containing ndhF change under different environmental stress conditions?

The NDH complex plays a crucial role in plant stress responses, with its activity modulated under various environmental conditions:

• Light intensity responses:

  • High light conditions typically increase NDH activity

  • This increase helps prevent over-reduction of the stroma

  • Upregulation of NDH genes has been observed under high light stress

• Temperature stress effects:

  • Heat stress generally enhances NDH-dependent cyclic electron flow

  • Cold stress can also increase NDH activity in some species

  • Temperature extremes appear to trigger increased reliance on NDH-mediated electron transport

• Drought and salinity impacts:

  • Water deficit conditions often increase NDH activity

  • Enhanced cyclic electron flow helps maintain pH gradient under drought

  • NDH activity correlates with tolerance to osmotic stress in many species

• Combined stress responses:

  • Multiple simultaneous stresses (e.g., drought + heat) show synergistic effects on NDH induction

  • The temporal dynamics of NDH activation vary depending on stress combination

  • Pre-exposure to certain stresses can prime NDH activity for subsequent stress events

Research has shown that the NDH complex helps prevent the overreduction of stroma, especially under stress conditions . This is achieved through its role in cyclic electron flow around PSI, which generates additional ATP without producing NADPH. The complex appears to be particularly important when plants face multiple or severe stresses that could otherwise lead to photoinhibition and oxidative damage.

What methodologies are most effective for studying the contribution of ndhF to cyclic electron flow in intact plants?

Studying the contribution of ndhF to cyclic electron flow in intact plants requires sophisticated approaches that preserve physiological conditions while enabling detailed measurements:

• Chlorophyll fluorescence techniques:

  • Post-illumination fluorescence rise (PIFR) measurements to quantify NDH activity

  • Pulse-amplitude modulation (PAM) fluorometry to assess cyclic electron flow

  • Fast chlorophyll fluorescence induction curves to evaluate electron transport chain function

  • Experimental protocol:

    • Dark-adapt leaves for 30 minutes

    • Apply saturating light pulse (3,000 μmol photons m^-2 s^-1) for 1 second

    • Measure post-illumination fluorescence rise in the subsequent dark period

    • Quantify the amplitude and kinetics of the PIFR as indicators of NDH activity

• P700 absorbance measurements:

  • Monitor P700 redox state changes

  • Measure PSI donor side limitation as an indicator of cyclic flow

  • Compare wild-type and NDH-deficient plants under different conditions

• Mutant comparison approaches:

  • Use knockout or knockdown lines for ndhF

  • Compare with PGR5-pathway mutants and double mutants

  • Assess photosynthetic parameters under various light and stress conditions

• Electrochromic shift (ECS) measurements:

  • Quantify proton motive force generation

  • Separate linear and cyclic electron flow contributions

  • Analyze the relationship between electron and proton transport

The most reliable results come from combining multiple approaches. For example, research has shown that double mutants lacking both NDH and PGR5-dependent pathways (like lhca6 pgr5) exhibit severe defects in growth and photosynthesis, particularly in mature leaves . This indicates the complementary roles of these two cyclic electron transport pathways and provides a methodology for assessing their relative contributions under different conditions.

How can recombinant ndhF be utilized to reconstruct an active NDH complex in vitro?

Reconstructing an active NDH complex in vitro represents one of the most challenging but potentially rewarding approaches to understanding this system:

• Component production strategy:

  • Express all necessary NDH subunits (including ndhF) as recombinant proteins

  • Consider co-expression systems for subunits that require interaction for stability

  • Purify components using affinity tags that can be removed without affecting activity

  • Optimize detergent and lipid environment for membrane protein reconstitution

• Assembly protocol development:

  • Investigate stepwise assembly of subcomplexes before final complex formation

  • Determine optimal protein:lipid ratios for reconstitution

  • Test various detergents and lipid compositions to mimic the thylakoid membrane environment

  • Consider nanodiscs or liposome incorporation for stability

• Activity verification methods:

  • Develop robust assays for NADH/NADPH oxidation

  • Monitor quinone reduction using either natural or artificial electron acceptors

  • Assess complex integrity through BN-PAGE or analytical ultracentrifugation

  • Characterize electron transfer kinetics using stopped-flow spectroscopy

• Structural characterization:

  • Apply single-particle cryo-EM to determine structure

  • Use hydrogen-deuterium exchange mass spectrometry to probe dynamics

  • Analyze subunit interactions through crosslinking mass spectrometry

What are the latest approaches for investigating the specific electron donor preference (NADH vs. NADPH) of pea NDH complex containing ndhF?

Determining the electron donor preference of the pea NDH complex remains an area of active research with some contradictory findings. Current advanced approaches include:

• Direct binding and kinetic studies:

  • Isothermal titration calorimetry (ITC) to measure binding affinities for both cofactors

  • Stopped-flow kinetics to determine reaction rates with NADH vs. NADPH

  • Enzyme kinetics comparing Km and Vmax values for both cofactors under identical conditions

  • Typical experimental setup:

    • Purified NDH complex: 1-5 μM

    • NADH/NADPH concentration range: 1-500 μM

    • Quinone acceptor: 100 μM

    • Temperature: 25°C

    • Buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl

• Structural approaches:

  • Site-directed mutagenesis of predicted nucleotide-binding sites

  • X-ray crystallography or cryo-EM of complex with bound cofactors

  • Computational docking and molecular dynamics simulations

  • Photoaffinity labeling with NAD(P)H analogues

• In organello validation:

  • Measurements in isolated intact chloroplasts

  • Selective inhibition of NADH or NADPH production pathways

  • Isotope labeling to track electron flow

Research findings show conflicting results regarding the preference for NADH versus NADPH. Some studies with purified pea NDH complex show it oxidizes NADH , while other studies suggest NADPH might be preferred in cyanobacterial NDH . Additionally, there are indications that ferredoxin might serve as a direct electron donor in certain contexts .

These contradictory findings suggest that the NDH complex might have evolved different cofactor preferences in different organisms, or that its activity might be regulated by factors not yet fully understood.

How can understanding ndhF function contribute to improving crop stress tolerance and photosynthetic efficiency?

Research on ndhF and the NDH complex has significant implications for crop improvement strategies:

• Enhancement of stress tolerance mechanisms:

  • Engineering improved NDH activity could boost plant performance under:

    • Drought conditions

    • High temperature stress

    • Fluctuating light environments

    • High salinity

  • Higher NDH activity correlates with better maintenance of photosynthesis under stress

• Photosynthetic efficiency optimization:

  • Fine-tuning cyclic electron flow can enhance ATP:NADPH production ratio

  • This balance is critical for carbon fixation efficiency

  • Modulating NDH activity might improve carbon assimilation under fluctuating conditions

  • Potential for up to 5-8% improvement in photosynthetic efficiency under certain conditions

• Integration with other photosynthetic enhancements:

  • Combining NDH improvements with Rubisco engineering

  • Coordinating with photorespiratory bypass approaches

  • Synergizing with chlorophyll antenna size adjustments

• Translational research approaches:

  • Screening germplasm collections for natural NDH variants

  • CRISPR-based editing of NDH-related genes

  • Marker-assisted breeding focusing on NDH complex components

  • Development of high-throughput phenotyping for NDH activity

Research has demonstrated that mutants completely lacking cyclic electron flow activity around PSI, such as crr pgr5 and lhca6 pgr5 double mutants, exhibit severe growth defects . This underscores the critical importance of these pathways for plant productivity and suggests potential targets for enhancement. By understanding the molecular details of ndhF function, researchers can develop targeted approaches to optimize NDH performance in crop plants, potentially improving both productivity and resilience.

What methods can be used to study the interactions between ndhF and other proteins in the thylakoid membrane?

Studying protein-protein interactions involving ndhF in the thylakoid membrane requires specialized approaches suitable for membrane proteins:

• Co-immunoprecipitation strategies:

  • Generate specific antibodies against ndhF

  • Use gentle solubilization methods to preserve native interactions

  • Identify interaction partners through mass spectrometry

  • Confirm interactions with reciprocal co-IPs

  • Experimental consideration:

    • Detergent selection is critical (e.g., digitonin, dodecyl maltoside)

    • Crosslinking prior to solubilization can stabilize transient interactions

    • Controls must include immunoprecipitation with non-specific antibodies

• Advanced imaging approaches:

  • Förster resonance energy transfer (FRET) with fluorescently tagged proteins

  • Bimolecular fluorescence complementation (BiFC) in chloroplasts

  • Super-resolution microscopy to visualize complex distribution and co-localization

  • Electron microscopy combined with immunogold labeling

• Chemical crosslinking coupled to mass spectrometry:

  • Apply membrane-permeable crosslinkers to intact thylakoids

  • Identify crosslinked peptides through specialized mass spectrometry approaches

  • Map interaction interfaces at the amino acid level

  • Build structural models based on crosslinking constraints

• Blue native PAGE with second dimension analysis:

  • Separate native complexes in the first dimension

  • Use denaturing conditions in the second dimension

  • Identify components through immunoblotting or mass spectrometry

  • Compare complex composition under different physiological conditions

Research using blue native PAGE has successfully identified the NDH-PSI supercomplex and its components. Mass spectrometry analysis of this complex detected subunits from both NDH and PSI, confirming their physical association. This approach revealed that the NDH-PSI supercomplex has a molecular mass of >1000 kDa , and identified two minor light-harvesting complex I proteins, Lhca5 and Lhca6, as required for the full-size NDH-PSI supercomplex formation.

What are the most promising techniques for real-time monitoring of ndhF-containing NDH complex activity in vivo?

Emerging techniques for real-time monitoring of NDH complex activity in living plants represent cutting-edge approaches in photosynthesis research:

• Advanced chlorophyll fluorescence imaging:

  • High-resolution spatial mapping of NDH activity across leaf surfaces

  • Time-resolved measurements capturing dynamic responses to environmental changes

  • Automated image analysis for quantitative assessment of NDH function

  • Implementation considerations:

    • Use modulated measuring light to avoid actinic effects

    • Apply far-red illumination to preferentially excite PSI

    • Measure post-illumination fluorescence rise as an indicator of NDH activity

    • Combine with gas exchange measurements for comprehensive analysis

• Genetically encoded fluorescent sensors:

  • Development of NADH/NADPH ratio sensors targeted to chloroplasts

  • Redox-sensitive fluorescent proteins fused to NDH complex components

  • FRET-based sensors that report on NDH conformational changes

  • Potential for tracking NDH activity at subcellular resolution

• Multi-wavelength absorption spectroscopy:

  • Simultaneous monitoring of P700, plastocyanin, and cytochrome redox states

  • Deconvolution of signals to isolate NDH-specific activity

  • Integration with other spectroscopic techniques for comprehensive assessment

• Field-deployable phenotyping platforms:

  • Adaptation of lab techniques for field conditions

  • High-throughput screening of germplasm for NDH function

  • Long-term monitoring under realistic environmental fluctuations

  • Correlation of NDH activity with crop performance metrics

These techniques are still in development, but they promise to provide unprecedented insights into NDH function in intact plants under natural conditions. The ability to monitor NDH activity non-destructively and in real-time will be particularly valuable for understanding its role in dynamic responses to environmental changes and for screening genetic variants with enhanced NDH function.

How might synthetic biology approaches be used to engineer novel functions into the ndhF subunit?

Synthetic biology offers exciting possibilities for engineering enhanced or novel functions into the ndhF subunit and the broader NDH complex:

• Cofactor specificity engineering:

  • Modify the nucleotide-binding domain to alter NADH/NADPH preference

  • Engineer bifunctional variants capable of utilizing both cofactors with high efficiency

  • Create variants with altered kinetic properties optimized for specific environmental conditions

  • Design approach:

    • Identify residues forming the nucleotide-binding pocket

    • Introduce mutations based on structural comparison with dehydrogenases of known specificity

    • Screen libraries using high-throughput activity assays

    • Validate promising candidates in vivo

• Environmental response tuning:

  • Engineer temperature-responsive variants for improved performance at temperature extremes

  • Develop redox-sensing domains that modulate activity based on cellular redox state

  • Create light-responsive elements that adjust NDH contribution under different light conditions

• Alternative electron transfer pathways:

  • Engineer direct electron transfer from ferredoxin to enhance cyclic electron flow

  • Create hybrid complexes incorporating features from both plant and cyanobacterial NDH

  • Develop variants that can participate in additional electron transport pathways

• Bioorthogonal labeling and control:

  • Incorporate unnatural amino acids for site-specific labeling

  • Develop optogenetic control of NDH activity

  • Create chemically-inducible systems for temporal control of NDH function