Recombinant Pisum sativum ATP synthase subunit c, chloroplastic (atpH)

What is the structure and function of ATP synthase subunit c in chloroplasts?

ATP synthase subunit c (atpH) is a critical component of the chloroplastic ATP synthase complex (CF₁Fₒ-ATP synthase). The c-subunit forms a ring structure in the membrane-embedded Fₒ domain, functioning as the proton-conducting rotor that drives ATP synthesis.

Structurally, each c-subunit folds into a hairpin of two transmembrane helices connected by a short partially structured loop. Multiple c-subunits (typically 14 in plant chloroplasts) assemble into a cylindrical ring structure. The c₁₄-ring has these key dimensions:

• Length: 60.32 Å

• Outer ring diameter: 52.30 Å

• Inner ring width: 40 Å

The c-ring works in concert with subunit a to form a proton channel. During photosynthesis, protons flow through this channel, causing the c-ring to rotate. This rotation is mechanically coupled to the F₁ sector, driving conformational changes that catalyze ATP synthesis .

High-resolution crystallographic studies (2.3 Å) of spinach chloroplast c-rings have revealed important features:

• A network of hydrogen bonds stabilizing the ring structure

• Intersubunit contacts that determine c-ring stoichiometry

• Circular electron densities inside the hydrophobic part of the internal pore, potentially representing bound lipids or other molecules

How do researchers determine the stoichiometry of c-rings in different species?

The stoichiometry of c-rings (number of c-subunits) varies across species from 8 to 15 subunits. This variation affects the H⁺-to-ATP ratio, a crucial bioenergetic parameter. Researchers employ several complementary techniques to determine c-ring stoichiometry:

Atomic Force Microscopy (AFM):
AFM studies have demonstrated that chloroplast ATP synthase c-rings contain 14 symmetrically distributed subunits that protrude from both membrane surfaces .

X-ray Crystallography:
Crystallization and diffraction analysis can definitively establish c-ring stoichiometry. For example, crystallographic studies of spinach chloroplast c-rings confirmed 14 subunits in the asymmetric unit cell .

Mass Spectrometry:
Mass spectrometry of intact c-rings can provide stoichiometric information based on the molecular weight of the complex.

Genetic Engineering Approach:
Researchers can confirm c-ring stoichiometry by genetic modification followed by functional analysis. For example, in a study where the native tobacco atpH gene was replaced with the sequence from Spirulina platensis (known to have a c₁₅-ring), the modified ATP synthase exhibited altered function consistent with a c₁₅-ring structure .

Experimental Protocol for Crystallographic Analysis:

• Isolate intact ATP synthase from chloroplasts

• Extract c-rings using detergent solubilization

• Purify by chromatography methods

• Crystallize using in meso crystallization techniques

• Collect X-ray diffraction data

• Determine structure and count number of subunits in the ring

What methods are most effective for recombinant expression of atpH from Pisum sativum?

Recombinant expression of chloroplastic atpH presents challenges due to its hydrophobic nature and membrane integration. Based on established protocols, the following methods have proven effective:

Expression Systems:

• E. coli: Most commonly used for atpH expression

• Yeast: Suitable for higher eukaryotic protein folding

• Baculovirus: Used for more complex expression needs

• Mammalian cells: For specific post-translational modifications

Expression Vectors and Tags:
Fusion with maltose-binding protein (MBP) has been successfully employed for spinach c-subunit expression, improving solubility and purification efficiency .

High-Yield Expression Protocol:

• Vector Construction:

  • Clone the atpH gene into an expression vector with an inducible promoter

  • Include a fusion tag to enhance solubility and purification

• Transformation and Culture:

  • Transform into an appropriate E. coli strain (BL21(DE3), C41(DE3), or C43(DE3))

  • Use high-cell-density methods to maximize yield

• Induction Strategies:

  • IPTG induction: Traditional approach with tight control

  • Autoinduction: Allows cultures to reach high cell density (OD₆₀₀: 10-20) before protein expression is automatically induced

• Expression Optimization:

  • Multiple small volume expressions (5 × 50 mL) instead of single large volume (250 mL)

  • Culture at lower temperatures (16-20°C) after induction to improve folding

  • Typical yields: 14-25 mg of labeled protein or 17-34 mg of unlabeled protein from 50 mL culture

This high-yield expression protocol can increase protein production by 9- to 85-fold compared to traditional methods .

How can researchers purify recombinant atpH protein to high homogeneity for structural studies?

Purifying membrane proteins like atpH requires specialized approaches. The following purification workflow has been successful for obtaining high-purity c-subunit:

Extraction and Solubilization:

• Harvest cells by centrifugation

• Resuspend in lysis buffer with protease inhibitors

• Disrupt cells using sonication, high-pressure homogenization, or enzymatic lysis with lysozyme (0.2 mg/ml, 37°C, 15 min)

• Isolate membranes by ultracentrifugation

• Solubilize with appropriate detergent (e.g., n-dodecyl-β-D-maltoside or LDAO)

Purification Strategy:
For tag-based purification (if using a fusion protein approach):

For Tag-less Approach:
A minimal two-step purification method has been developed that may be applicable:

• Buffer exchange using tangential flow filtration

• Q-membrane filtration to remove impurities
  This approach can yield 1-2 mg of protein with >60% purity, sufficient for many biophysical characterizations .

Quality Control:

• SDS-PAGE: Assess purity (target: >85% as determined by densitometry)

• Mass spectrometry: Confirm identity and detect modifications

• Circular dichroism: Verify proper alpha-helical secondary structure

What are the challenges in crystallizing the c-ring of chloroplastic ATP synthase?

Crystallizing membrane protein complexes like the c-ring presents specific challenges that researchers must overcome:

Major Challenges:

• Detergent Management: Finding detergents that maintain c-ring integrity while allowing crystal formation

• Stability Issues: Maintaining the oligomeric state during purification and crystallization

• Low Protein Yields: Obtaining sufficient quantities of pure, homogeneous protein

• Crystal Packing: Achieving well-ordered crystals with minimal detergent-filled voids

Successful Crystallization Approach:
Researchers have successfully obtained high-resolution (2.3 Å) crystals of spinach chloroplast c₁₄-rings using in meso crystallization:

• Starting Material: Begin with intact, active chloroplast F₁F₀-ATP synthase

• Extraction Method: Selective extraction of the c-ring while maintaining its oligomeric state

• Crystallization Conditions:

  • Crystal type: Type I crystals with plate-like shape

  • Crystal size: Up to 20 µm

  • Space group: I121

  • Cell parameters: a = 93.14 Å, b = 96.34 Å, с = 158.68 Å, α = γ = 90° and β = 106.72°

  • Incubation time: Approximately 2 months

Alternative Approaches:

• Lipidic cubic phase (LCP) crystallization

• Reconstitution into nanodiscs prior to crystallization

• Cryo-EM analysis as an alternative to crystallography

Notably, researchers used a biochemically characterized preparation of the complete chloroplast F₁F₀-ATP synthase that showed high purity by SDS-PAGE and BN-PAGE, with the c-subunit migrating as an intact oligomer. The preparation maintained ATP synthesis activity (41±4 ATP per enzyme per second) after reconstitution into liposomes .

How does genetic modification of the atpH gene affect ATP synthase function and photosynthesis?

Genetic modification of atpH provides insights into structure-function relationships of the ATP synthase. Research on engineered c-rings has revealed fascinating adaptations:

Modification Approaches:

• Gene Replacement: Substituting native atpH with variants from other species

• Site-Directed Mutagenesis: Altering specific residues involved in proton conduction or ring stability

• Promoter Modification: Altering expression levels of atpH

Case Study: c₁₄ to c₁₅ Ring Conversion:
In a landmark study, researchers replaced the tobacco atpH gene with a modified version based on Spirulina platensis (which naturally has a c₁₅-ring). The results revealed remarkable physiological adaptations:

The transplastomic plants with c₁₅-rings maintained normal photosynthetic growth despite the drastically reduced ATP synthase content. They accomplished this by increasing the membrane potential component of the proton motive force, ensuring sufficient proton flux through the c₁₅-ring without inducing low pH-induced feedback inhibition of electron transport .

Methodology for atpH Modification:

• Design synthetic atpH gene with desired modifications

• Insert into a chloroplast transformation vector with selectable marker

• Perform biolistic transformation of chloroplasts

• Select for transformants and confirm homoplasmy

• Analyze ATP synthase content by immunoblotting

• Measure photosynthetic parameters and growth

This research demonstrates the remarkable adaptability of the photosynthetic apparatus to alterations in a key bioenergetic parameter.

What techniques are used to study the proton pathway through the ATP synthase c-ring?

Understanding the proton pathway through ATP synthase is crucial for elucidating its mechanism. Researchers employ several complementary techniques:

Structural Approaches:

• Cryo-EM: High-resolution (2.9-3.4 Å) cryo-EM structures of complete chloroplast ATP synthase have revealed the proton pathway to and from the c-ring. This technique has successfully resolved sidechains of all 26 protein subunits .

• X-ray Crystallography: Crystal structures of c-rings at high resolution (2.3 Å) show essential features of the proton-binding sites .

Spectroscopic Methods:

• Electrochromic Pigment Absorbance Shift (ECS): This non-invasive technique measures changes in green light absorption by photosynthetic pigments to monitor electrical gradients across thylakoid membranes.

• Light Scattering (LS): Used to measure proton gradients and pH changes in chloroplasts.

Experimental Protocol for Proton Gradient Measurement:

• Sample Preparation: Use intact leaves or leaf segments

• ECS Measurement: Record absorption changes at specific wavelengths (typically ~520 nm)

• Analysis of Components:

  • ECS pmf: Measures the total proton motive force

  • ECS ΔpH: Measures the proton gradient component

  • ECS ΔΨ: Measures the electrical potential component

• H⁺-ATP Synthase Activity: Determined from ECS relaxation kinetics

Key Findings:
A study on pea (Pisum sativum) leaves demonstrated that variation potential (VP, an electrical signal) decreased the rate constant of ECS relaxation, reflecting reduced proton conductivity of H⁺-ATP synthase. This effect was similar to artificially suppressing photosynthesis by lowering CO₂ concentration, suggesting a regulatory mechanism linking electrical signaling to ATP synthesis .

Measurement of ATP Synthase Activity:
Researchers can quantify ATP synthase activity by:

• Reconstituting purified ATP synthase into liposomes

• Applying an electrochemical proton gradient

• Measuring ATP synthesis rates (e.g., 41±4 ATP per enzyme per second for spinach chloroplast ATP synthase)

How does the redox regulation mechanism of chloroplastic ATP synthase function?

Chloroplast ATP synthase has evolved a unique redox regulation mechanism that prevents wasteful ATP hydrolysis in the dark. This regulation involves a specific structural element in the γ-subunit:

Redox Switch Structure:

• The chloroplast γ-subunit contains a conserved ~40 amino acid insertion not found in bacterial or mitochondrial homologs

• This insertion forms two β-hairpins arranged in an L shape

• Located between the nucleotide-free β subunit (βempty) and the γ-rotor

• Contains redox-active cysteines that can form a disulfide bond

Mechanism of Action:

• In the Dark (Oxidizing Conditions):

  • The γ-subunit hairpin forms a disulfide bond

  • This creates a β-hairpin "chock" that physically blocks rotation

  • ATP hydrolysis is prevented, conserving energy when photosynthesis is inactive

• In the Light (Reducing Conditions):

  • Reducing equivalents from photosynthesis break the disulfide bond

  • The inhibitory conformation is released

  • Rotation is permitted, allowing ATP synthesis

Research Methods to Study Redox Regulation:

• Site-Directed Mutagenesis: Modifying the cysteine residues to prevent disulfide formation

• Structural Analysis: Using cryo-EM to visualize the conformational changes

• Activity Assays: Measuring ATP synthesis/hydrolysis under different redox conditions

Cryo-EM studies at 2.9-3.4 Å resolution have directly visualized this regulatory mechanism, showing how the redox-controlled γ-subunit insertion physically blocks rotation in oxidizing conditions .

Physiological Significance:
This elegant regulation mechanism ensures that:

• ATP synthesis occurs efficiently during photosynthesis

• ATP is not wastefully hydrolyzed in the dark

• Energy conservation is maintained during diurnal cycles

This represents a plant-specific adaptation that is not present in bacterial or mitochondrial ATP synthases.

What methods are most effective for analyzing c-ring assembly and oligomerization?

Assessing proper assembly of recombinant atpH into functional c-rings requires specialized techniques:

Electrophoretic Methods:

• Blue Native PAGE (BN-PAGE): Preserves native protein complexes during electrophoresis

  • Sample preparation: Solubilize membranes with mild detergents

  • Analysis: Intact c-rings migrate as high molecular weight bands

  • Validation: The presence of predominantly intact CF₁F₀-ATP synthase (>500 kDa) with minimal unbound CF₁ indicates proper assembly

• SDS-PAGE with Sample Preparation Variations:

  • Without boiling: Intact c-rings often remain associated despite SDS treatment

  • With boiling: Complete dissociation into monomers for stoichiometry estimation

  • Semi-native conditions: Can reveal intermediate states of assembly

Microscopy Techniques:

• Atomic Force Microscopy (AFM): Enables visualization of c-ring topology and subunit arrangement

• Electron Microscopy: Single-particle analysis can resolve subunit organization

Functional Assays:

• Reconstitution into Liposomes:

  • Incorporate purified c-rings into artificial membranes

  • Generate a proton gradient

  • Measure proton conductance

• ATP Synthesis Activity:

  • Reconstitute entire ATP synthase into liposomes

  • Apply electrochemical proton gradient

  • Measure ATP synthesis (e.g., spinach ATP synthase: 41±4 ATP per enzyme per second)

Experimental Protocol for Assessing c-Ring Integrity:

• Isolate membranes containing ATP synthase

• Solubilize with mild detergent (1% digitonin)

• Separate by BN-PAGE

• Perform in-gel ATPase activity assay to confirm functional assembly

• For higher resolution analysis, extract bands and analyze by mass spectrometry

How does the membrane environment affect c-ring stability and function?

The lipid environment plays a crucial role in c-ring stability and function. Recent research has revealed fascinating aspects of this protein-lipid interaction:

Key Lipid-Protein Interactions:

• Internal Lipid Pore: High-resolution (2.3 Å) structures of c-rings from spinach chloroplasts revealed circular electron densities inside the hydrophobic part of the internal pore. These densities might represent isoprenoid quinones (such as plastoquinone in chloroplasts) .

• Lipid Binding Sites: Specific lipid binding sites have been identified at the interface between c-subunits, contributing to ring stability.

Experimental Approaches to Study Membrane Effects:

• Detergent Screening:
  Different detergents can be systematically tested for their ability to maintain c-ring integrity during purification:

  • Mild detergents: digitonin, DDM

  • Medium strength: LDAO, C₁₂E₈

  • Harsh detergents: SDS, Triton X-100

• Reconstitution Studies:
  Purified c-rings can be reconstituted into:

  • Liposomes of defined lipid composition

  • Nanodiscs with controlled lipid environment

  • Native thylakoid membrane lipid extracts

• Biophysical Characterization:

  • Differential scanning calorimetry (DSC) to measure thermal stability

  • Circular dichroism (CD) to assess secondary structure

  • Fluorescence spectroscopy to monitor conformational changes

Research Findings:

• The c-ring shows unusual structural features in its hydrophobic core, with electron densities arranged in circles parallel to the membrane plane.

• These features appear universal among ATP synthases from various species (archaea, bacteria, and eukaryotes).

• Spectroscopic evidence suggests these may be isoprenoid quinones (like plastoquinone in chloroplasts).

• The large distance between polar/apolar interfaces inside the c-ring (unusually wide hydrophobic region) supports this hypothesis .

This finding suggests a potential universal role for quinone-like molecules as cofactors in ATP synthases, possibly stabilizing the c-ring and preventing ion leakage through it.

What are the most reliable antibodies and immunological tools for studying atpH?

Researchers studying atpH require reliable immunological tools. The following antibodies and methods have been validated for atpH detection:

Commercial Antibodies:

• Anti-AtpH (AS05 071):

  • Type: Polyclonal

  • Host: Rabbit

  • Reactivity: Arabidopsis thaliana, Spinacia oleracea, Nicotiana benthamina, Thermosynechococcus elongatus

  • Predicted reactivity: Algae, Cannabis sativa, Cyclotella cryptica, Glycine max, Oryza sativa, Physcomitrium patens, Phaeodactylum tricornutum, Pisum sativum, Populus alba, Pinus thunbergii, Thalassiosira pseudonana, Zea mays, Vitis vinifera

  • Format: Lyophilized serum (200 μl)

  • Reconstitution: Add 200 μl of sterile water

  • Storage: -20°C (make aliquots after reconstitution)

  • Applications: Western blot

  • Recommended dilution: 1:1000-1:10,000

Immunodetection Protocol:

• Sample Preparation:

  • For purified ATP synthase: Load 0.7-15 μg protein

  • For thylakoid preparations: Load 40-50 μg protein

  • Separate on 12% polyacrylamide gel

  • Transfer to PVDF membrane

• Western Blot Procedure:

  • Block membrane: 0.5 hours

  • Primary antibody: Anti-AtpH at 1:1000 dilution, 1 hour

  • Secondary antibody: Anti-rabbit-HRP at 1:5000 dilution, 1.25 hours

  • Detection: Chemiluminescence reagent

  • Expected molecular weight: 8 kDa (for Arabidopsis thaliana)

Detection Sensitivity:
The antibodies can detect both the monomeric c-subunit and intact c-rings in properly prepared samples, making them versatile tools for studying both the expression levels and assembly state of ATP synthase.

How can researchers study the energetics of proton transport through the c-ring?

The energetics of proton transport through the c-ring is fundamental to understanding ATP synthesis. Researchers employ several approaches to study this process:

Biophysical Techniques:

• Electrochromic Shift (ECS) Measurements:

  • Based on absorption changes in photosynthetic pigments

  • Provides real-time monitoring of electrical gradients

  • Components measured:

    • ECS pmf: Total proton motive force

    • ECS ΔpH: Proton gradient component

    • ECS ΔΨ: Electrical potential component

• ECS Relaxation Kinetics:

  • Dark relaxation of the ECS signal after light-to-dark transition

  • Rate constant (kECS) directly relates to H⁺-ATP synthase activity

  • Can be used to quantify changes in ATP synthase function

Experimental Protocol for ECS Measurements:

• Use intact leaves or leaf segments (e.g., 14-21 day-old pea seedlings)

• Monitor absorption changes following light-to-dark transitions

• Fit relaxation kinetics to determine rate constants

• Compare under different conditions (e.g., normal vs. low CO₂)

Energetic Calculations:
The free enthalpy (ΔG) of ATP hydrolysis under physiological conditions in chloroplasts is approximately -51 kJ/mol. For a c₁₄-ring in chloroplasts:

• Each proton contributes -51×3/14 or -10.9 kJ/mol to ATP synthesis

• The three observed rotary states contribute energy differences of -43.7, -48.1, and -61.2 kJ/mol per step

Key Research Findings:

• Unequal Energy Distribution:
  Surprisingly, the three conformations of chloroplast ATP synthase are separated by rotations of 103°, 112°, and 145° (or 4, 4.4, and 5.6 c-subunits), rather than the expected equal distributions .

• Peripheral Stalk Elasticity:
  The peripheral stalk (subunits b and b') bends relative to the central axis, coupling F₁ elastically to F₀. This acts like an elastic spring, evening out energy differences between rotational states to optimize ATP synthesis .

• Regulation by Electrical Signals:
  Studies in pea leaves showed that variation potential (an electrical signal) decreased H⁺-ATP synthase activity by reducing the rate constant of ECS relaxation. This effect was similar to artificially suppressing photosynthesis by lowering CO₂ concentration .

These approaches have revealed sophisticated energy distribution and regulatory mechanisms that optimize ATP synthesis under varying conditions.

What strategies exist for increasing recombinant atpH protein yields?

Producing high yields of recombinant atpH protein requires specialized strategies to overcome challenges associated with membrane protein expression:

Expression System Optimization:

• High-Cell-Density Methods:

  • Start with rich medium (LB, 2× YT) to achieve high initial cell density (OD₆₀₀ of 3-7)

  • Switch to minimal medium before induction

  • Culture at optimized temperature for 1-1.5 hours

  • Induce with IPTG

  • Final cell density can reach OD₆₀₀ of 10-20

  • Results in 9- to 85-fold enhancement in protein yields

• Autoinduction Approach:

  • Uses media containing glucose, lactose, and glycerol

  • Glucose initially prevents induction

  • As glucose is depleted, lactose induces protein expression

  • Allows cultures to reach high density before induction begins

  • Typical yields: 14-25 mg of labeled proteins from 50-mL culture

Expression Protocol Refinements:

• Multiple small-volume expressions (5 × 50 mL) instead of single large-volume (250 mL)

• Lower post-induction temperatures (16-20°C) to improve protein folding

• Codon optimization for the expression host

• Co-expression with chaperones to assist folding

Fusion Tags to Enhance Solubility:

• Maltose-binding protein (MBP) fusion has been successfully used for spinach c-subunit expression

• Other potential fusion partners: Thioredoxin, SUMO, GST

• Cleavable tags allow removal after purification

Extraction Optimization:
Different extraction methods can be employed based on the expression level and experimental goals:

By combining these strategies, researchers have achieved yields of 17-34 mg of unlabeled protein from 50-mL cultures, providing sufficient material for structural and functional studies .

How do mutations in the atpH gene affect ATP synthase assembly and function?

Mutations in the atpH gene provide valuable insights into structure-function relationships of ATP synthase. Research has revealed several important aspects:

Complete Gene Deletion:
When the entire atpH gene was replaced with a selectable marker (aadA cassette) in tobacco chloroplasts, plants were unable to grow without acetate supplementation. This confirms the essential nature of the c-subunit for photosynthetic ATP synthesis .

c-Ring Stoichiometry Modification:
By converting the conserved glycine-rich motif in tobacco atpH from GLAVGLASIGPGVGQGT to aLAVGigSIGPGlGQGq (modeled after Spirulina platensis), researchers created plants with a c₁₅-ring instead of the normal c₁₄-ring. Key findings included:

• ATP Synthase Content: Reduced to approximately 25% of wild-type levels

• Subunit Composition: Levels of various ATP synthase subunits (AtpH, AtpF, AtpA, AtpB) were all decreased to less than 50% of wild-type levels

• Growth: Despite reduced ATP synthase content, plants grew normally

• Photosynthetic Electron Transport: Remained unaffected

• Bioenergetic Adaptation: Increased contribution of membrane potential to proton motive force compensated for the altered H⁺/ATP ratio

Molecular Basis of c-Ring Assembly:
The sequence surrounding the glycine repeats is highly conserved in angiosperms and critical for proper c-ring assembly. The interface between c-subunits contains a network of hydrogen bonds that determines correct oligomerization .

Experimental Approach for Studying atpH Mutations:

• Vector Construction:

  • Design mutations in the atpH gene

  • Clone into vector containing selectable marker (e.g., aadA cassette)

  • Include suitable flanking sequences for homologous recombination

• Transformation:

  • Biolistic transformation of chloroplasts

  • Selection on medium with antibiotic

  • Verification of homoplasmy by DNA hybridization

• Analysis:

  • Immunoblotting to quantify ATP synthase subunits

  • BN-PAGE to assess complex assembly

  • Physiological measurements (growth, photosynthesis)

  • Biophysical characterization (membrane potential, proton gradient)

This research demonstrates that while the c-subunit is essential, plants can adapt to significant changes in ATP synthase content and c-ring stoichiometry through remarkable compensatory mechanisms.

How can researchers use atpH as a genetic marker for chloroplast DNA analysis?

The atpH gene has valuable applications as a genetic marker for plant identification and molecular taxonomy studies:

Advantages of atpH as a Genetic Marker:

• Location within the large single copy region of the chloroplast genome

• Suitable rate of sequence evolution for species discrimination

• Conserved regions for primer design flanking variable regions

• Successful amplification from DNA of varied quality and concentration

Applications in Plant Research:
The chloroplast atpF-atpH intergenic region has been effectively used for:

• Molecular systematic studies in plants

• DNA barcoding for species identification

• Phylogenetic analysis and molecular taxonomy

Experimental Methodology:

• DNA Extraction:

  • Extract DNA from silica-dried leaves

  • Assess quality by spectrophotometry (OD 260/280 ratio)

• PCR Amplification:

  • Use primers targeting the atpF-atpH intergenic region

  • PCR efficiency may be affected by DNA template quality (OD 260/280 ratio below 1.54 may result in poor amplification)

  • Successful amplification reported for 91.6% of Anthurium species DNA samples using atpF-atpH primer pairs

• Sequence Analysis:

  • Sequence PCR products

  • Align sequences for comparison

  • Identify polymorphisms for species discrimination

Performance Comparison:
When compared with other chloroplast markers (trnH-psbA, psbK-psbI, rpoB, rpoC1, matK, rbcL), the atpF-atpH region has shown good amplification success and suitable variation for species discrimination .

This application demonstrates how a fundamental component of the photosynthetic machinery also serves as a valuable tool for plant evolutionary studies and biodiversity assessment.