Recombinant Oryza nivara ATP synthase subunit c, chloroplastic (atpH)

What is the structural and functional role of ATP synthase subunit c in chloroplasts?

ATP synthase in chloroplasts produces adenosine triphosphate (ATP) required for photosynthetic metabolism. The synthesis of ATP is mechanically coupled to the rotation of a ring of c-subunits embedded in the thylakoid membrane . This c-subunit ring (designated as cn) plays a critical role in energy transduction by facilitating proton translocation across the thylakoid membrane along an electrochemical gradient .

The rotation of the c-subunit ring is directly coupled to the rotation of the γ-stalk in the F1 region, where subunit γ functions as a shaft inside the α3β3 head. This mechanical coupling drives the catalysis of the ADP + Pi → ATP reaction occurring at each of the three α-β subunit interfaces in F1 . This cyclical sequence of rotation, proton translocation and catalysis produces 3 ATP molecules for every n protons that pass from the lumen to the stroma, where n represents the number of c-subunits in the ring .

In chloroplasts, ATP synthase is classified as an F-type enzyme, which can operate reversibly – either synthesizing ATP when protons flow down their gradient or pumping protons when ATP is hydrolyzed . The chloroplastic ATP synthase fulfills the crucial function of harnessing the proton motive force generated during the light reactions of photosynthesis to produce ATP for carbon fixation and other metabolic processes.

How does the atpH gene in Oryza nivara compare to its homologs in other plant species?

The atpH gene encodes subunit c (also called subunit III) of the ATP synthase in chloroplasts. While the search results don't specifically address atpH in Oryza nivara, comparative analysis can be inferred from data on other plant species. The atpH gene is part of the chloroplast genome, as illustrated by the mapping and sequencing of this gene in Spinacia oleracea (spinach) .

In spinach, the c-subunit consists of 81 amino acids (UniProtKB accession no.: P69447) . Given the conserved nature of chloroplast genes across plant species, the Oryza nivara atpH gene likely encodes a protein of similar length, though species-specific variations may exist in certain amino acid positions. These variations might contribute to differences in ATP synthase efficiency or environmental adaptations.

The structure of the atpH gene appears relatively simple, encoding a small hydrophobic protein that forms α-helices embedded in the thylakoid membrane . When studying Oryza nivara atpH, researchers should consider both the high degree of conservation typical of chloroplast-encoded proteins and the potential species-specific adaptations that might be present in rice as compared to other plant species like spinach or Arabidopsis.

What techniques are available for identifying and characterizing the atpH gene product in Oryza nivara?

Several techniques can be employed to identify and characterize the atpH gene product (subunit c) in Oryza nivara. Based on approaches used for other plant species, researchers can implement a comprehensive strategy combining molecular biology, biochemistry, and structural biology methods.

For protein identification, immunoblotting (Western blotting) using antibodies specific to the c-subunit can confirm the expression and approximate molecular weight of the protein . In published research, 12% polyacrylamide gels have been successfully used for separating the c-subunit before immunoblotting .

Mass spectrometry provides more detailed characterization, allowing precise determination of the molecular weight and potential post-translational modifications. For structural characterization, circular dichroism spectroscopy can confirm the expected α-helical secondary structure of the purified c-subunit, as demonstrated in studies with spinach c-subunit .

Functional characterization can employ techniques such as the 9-amino-6-chloro-2-methoxyacridine (ACMA) assay, which measures ATP synthase enzymatic rate using the reverse mode (ATP hydrolysis driving proton translocation) . This assay requires preparation of submitochondrial vesicles (SMVs) or similar membrane vesicles from chloroplasts .

What are the optimal strategies for recombinant expression of Oryza nivara ATP synthase subunit c?

The recombinant expression of hydrophobic membrane proteins like ATP synthase subunit c presents significant challenges. Based on successful approaches with spinach chloroplast ATP synthase c-subunit, several strategies can be considered for Oryza nivara atpH.

Codon optimization represents an essential first step when designing a synthetic gene for expression in E. coli or other heterologous systems. For spinach c-subunit expression, researchers successfully designed a synthetic atpH gene with codons optimized for E. coli expression, with terminal restriction sites added for cloning . This approach can be directly applied to Oryza nivara atpH, using software such as Gene Designer to select optimal codons for the expression host .

The choice of expression vector and fusion tag significantly impacts success rates. Comparative testing of multiple vector constructs is advisable. For spinach c-subunit, vectors tested included pMAL-c2x (for maltose-binding protein fusion), pET-32a(+), and pFLAG-MAC, with pMAL-c2x showing promising results . These same vector systems could be tested for Oryza nivara c-subunit expression.

Co-expression with chaperone proteins has proven valuable for improving yields of difficult-to-express membrane proteins. The co-transformation of E. coli with both the expression vector and a plasmid expressing chaperone proteins DnaK, DnaJ, and GrpE (such as pOFXT7KJE3) can substantially increase yields of recombinant proteins that are toxic or otherwise challenging to produce .

Induction conditions require careful optimization, with parameters such as temperature, IPTG concentration, and induction duration being critical variables. For spinach c-subunit, induction with 1.0 mM IPTG for 30 minutes proved effective . Similar optimization studies would be necessary for Oryza nivara c-subunit expression.

How does c-ring stoichiometry in Oryza nivara ATP synthase impact the proton-to-ATP ratio and photosynthetic efficiency?

The number of c-subunits per ring (n) in ATP synthase varies among organisms, with documented values ranging from c10 to c15 . This variability directly determines the coupling ratio of ions transported to ATP generated, which ranges from 3.3 to 5.0 among studied organisms . The coupling ratio is calculated as n/3, since 3 ATP molecules are generated per complete rotation of the c-ring, regardless of the number of c-subunits it contains .

The exact purpose or evolutionary advantage of varying c-ring stoichiometries remains undefined, despite several hypotheses . Some theories suggest adaptations to different environmental niches, with variations in required ATP output or available proton motive force. Comparative studies between Oryza nivara and other plant species could provide insights into whether rice has evolved specific adaptations in its ATP synthase stoichiometry related to its semi-aquatic growth environment.

Understanding the factors influencing c-ring stoichiometry in Oryza nivara would contribute to broader knowledge about the relationship between ATP synthase structure and plant bioenergetics. The successful recombinant expression of c-subunit could enable experiments aimed at reconstituting the c-ring in vitro and investigating the determinants of its assembly and stoichiometry .

What role might ATP synthase subunit c play in chloroplast-to-nucleus retrograde signaling in Oryza nivara?

Chloroplast-to-nucleus (retrograde) signaling is essential for coordinating nuclear gene expression with chloroplast function and development. While ATP synthase subunit c has not been directly implicated in retrograde signaling pathways, the ATP synthase complex's central role in energy metabolism suggests potential indirect involvement.

Research with Arabidopsis thaliana has identified biogenic chloroplast-to-nucleus signaling pathways, including signals derived from tetrapyrrole synthesis, singlet oxygen (1O2) production, and chloroplast gene expression . Disturbances in ATP synthesis might impact these pathways by altering chloroplast energy status or redox state.

The chloroplast has been implicated as a sensor for many environmental signals that must be conveyed to the nucleus . The ATP synthase, as a key energy-transducing complex, could be involved in sensing energy status changes that trigger retrograde signals. For instance, changes in the redox state of electron carriers can regulate transcription of genes encoding components of photosystems in chloroplasts .

In Arabidopsis, chloroplast sensor kinase (CSK) exhibits redox-dependent auto-phosphorylation and influences the expression of photosystem genes in response to light-induced changes in plastoquinone redox state . Similar mechanisms might exist involving ATP synthase components, including subunit c, in Oryza nivara, potentially linking energy status to gene expression.

Exploring the potential role of ATP synthase subunit c in retrograde signaling would require experiments examining how mutations or altered expression of atpH affects nuclear gene expression patterns, particularly under conditions that challenge chloroplast function.

What purification methods are most effective for isolating recombinant Oryza nivara ATP synthase subunit c?

Purifying hydrophobic membrane proteins like ATP synthase subunit c requires specialized techniques that maintain protein stability while removing contaminants. Based on effective methods for spinach c-subunit, several approaches can be recommended for Oryza nivara c-subunit purification.

Fusion protein strategies significantly enhance purification efficiency. The maltose-binding protein (MBP) fusion approach has proven successful for spinach c-subunit . In this method, the synthetic atpH gene is cloned into a vector like pMAL-c2x to create an MBP-c-subunit fusion protein, which improves solubility and provides an affinity purification tag . After expression in E. coli, the fusion protein can be purified using amylose resin chromatography .

After initial affinity purification, additional chromatography steps may be necessary to achieve high purity. Size exclusion chromatography can separate the target protein from contaminating proteins of different molecular weights, while ion exchange chromatography can provide further purification based on charge differences .

For membrane proteins like subunit c, detergent selection is critical. Appropriate detergents must effectively solubilize the protein from membranes while maintaining native structure. Detergents such as n-dodecyl β-D-maltoside (DDM) or n-octyl β-D-glucopyranoside (OG) are commonly used for membrane protein purification and could be tested for Oryza nivara c-subunit .

Purity assessment should employ multiple methods, including SDS-PAGE with appropriate percentage gels (12% polyacrylamide has been effective for c-subunit visualization), Western blotting with c-subunit specific antibodies, and potentially mass spectrometry for definitive identification .

How can researchers functionally characterize recombinant Oryza nivara ATP synthase subunit c?

Functional characterization of recombinant ATP synthase subunit c requires specialized assays addressing both its structural integrity and its ability to form functional c-rings or integrate into the complete ATP synthase complex.

Secondary structure analysis via circular dichroism (CD) spectroscopy can confirm that the purified recombinant protein maintains the expected α-helical structure characteristic of ATP synthase c-subunits . This represents an essential first validation that the recombinant protein is properly folded.

Reconstitution assays can test the ability of recombinant c-subunit to assemble into oligomeric rings, a critical feature for its biological function. Such reconstitution might be performed in artificial liposomes or nanodiscs that mimic the native membrane environment . The assembled c-rings can then be visualized using techniques like transmission electron microscopy with negative staining or atomic force microscopy.

Functional assays for ATP synthase activity require either reconstitution of the complete ATP synthase complex or integration of the recombinant c-subunit into c-subunit depleted ATP synthase preparations. The ACMA assay mentioned previously measures proton translocation driven by ATP hydrolysis and could be adapted to test recombinant c-subunit functionality .

Electrophysiological approaches provide another functional assessment method. Patch-clamp recordings of membrane vesicles containing reconstituted c-rings can detect the presence of ion channels or leaks associated with the c-subunit . These measurements can reveal whether the recombinant protein forms channels with conductance properties similar to those observed in native systems.

What experimental approaches can determine if Oryza nivara ATP synthase subunit c contributes to ion leakage across thylakoid membranes?

Recent research suggests that ATP synthase c-subunits can form or contribute to ion leak channels across membranes, affecting membrane efficiency and potentially cellular metabolism . Several experimental approaches can determine if Oryza nivara c-subunit exhibits similar properties.

Electrophysiological measurements provide direct evidence of channel activity. Using patch-clamp techniques on membrane vesicles containing reconstituted c-subunit rings can detect ion conductance across the membrane . In studies of mitochondrial ATP synthase, researchers observed that the c-subunit ring can form channels of varying conductances that contribute to membrane leakage .

Membrane potential assays using fluorescent dyes sensitive to membrane potential can measure ion leakage indirectly. In such assays, vesicles containing reconstituted c-subunits would be loaded with potential-sensitive dyes, and changes in fluorescence would indicate dissipation of membrane potential due to ion leakage .

Pharmacological approaches with known modulators of ATP synthase channels can help characterize the c-subunit leak properties. Compounds like dexpramipexole (Dex) have been shown to reduce conductance through the ATP synthase leak channel in patch-clamp recordings . Testing such compounds on Oryza nivara c-subunit could reveal similar regulatory mechanisms.

Mutagenesis studies targeting specific residues in the c-subunit can identify amino acids critical for channel formation or gating. Mutant versions of the recombinant c-subunit could be tested in the aforementioned assays to determine their impact on channel activity, providing insights into the structural basis of ion leakage.

How can studies of recombinant Oryza nivara ATP synthase subunit c inform crop improvement strategies?

Understanding the structure, function, and regulation of ATP synthase subunit c in Oryza nivara has potential applications for crop improvement, particularly in areas related to photosynthetic efficiency and stress tolerance.

The c-ring stoichiometry directly influences the energetic efficiency of ATP production, affecting the number of protons required per ATP synthesized . Comparative studies between different rice varieties or between rice and other crop species could reveal whether natural variations in ATP synthase efficiency correlate with agronomically important traits such as photosynthetic rate or biomass production.

Environmental stress responses often involve changes in energy metabolism. Since ATP synthase is central to energy production, understanding how its components, including subunit c, respond to stresses like drought, heat, or high light could provide insights into stress adaptation mechanisms. Research in Arabidopsis has shown connections between chloroplast signaling pathways and heat and drought stress responses , suggesting similar connections might exist in rice.

Genetic engineering approaches targeting ATP synthase subunit c could potentially enhance photosynthetic efficiency or stress tolerance. For instance, modifications that optimize c-ring stoichiometry for specific environmental conditions might improve energy conversion efficiency. Such approaches would require thorough understanding of the relationship between c-subunit sequence, ring assembly, and functional properties, which studies of recombinant subunit c could provide.

Comparative analysis between wild rice species like Oryza nivara and cultivated rice (Oryza sativa) could reveal evolutionary adaptations in ATP synthase that might be valuable for crop improvement. Wild rice species often possess valuable traits for stress tolerance that have been lost during domestication, and these might include optimizations in energy metabolism components.

What methods can be used to study the integration of recombinant subunit c into intact chloroplasts?

Studying the integration of recombinantly produced ATP synthase subunit c into intact chloroplasts presents technical challenges but offers valuable insights into chloroplast biogenesis and ATP synthase assembly.

Chloroplast transformation systems provide one approach for introducing recombinant subunit c directly into the chloroplast genome. While technically challenging, chloroplast transformation has been established for rice and could be used to introduce tagged or modified versions of atpH . This approach would ensure that the recombinant protein is expressed within the chloroplast, where it would normally be synthesized.

In vitro chloroplast import assays offer an alternative approach for studying the integration of nuclearly-encoded recombinant proteins. Although ATP synthase subunit c is chloroplast-encoded in wild-type plants, recombinant versions with appropriate transit peptides could be designed for import studies. This approach could reveal insights about membrane protein integration mechanisms even though it doesn't represent the natural synthesis pathway.

Co-immunoprecipitation and proximity labeling techniques can identify interaction partners of recombinant subunit c during assembly. By introducing tagged versions of the protein and then isolating protein complexes that contain it, researchers can determine which other ATP synthase components interact with subunit c during the assembly process.

How do variations in ATP synthase subunit c impact photosynthetic efficiency under different environmental conditions?

ATP synthase performance directly influences photosynthetic efficiency, and understanding how variations in subunit c affect this relationship under different environmental conditions could provide valuable insights for both basic science and agricultural applications.

Temperature effects on ATP synthase efficiency could be particularly relevant for crop adaptation to climate change. The c-ring structure and assembly might be temperature-sensitive, affecting proton conductance or ATP synthesis rates at temperature extremes. Studies with recombinant Oryza nivara subunit c under varying temperature conditions could reveal adaptation mechanisms specific to rice.

Light intensity fluctuations require rapid adjustments in photosynthetic machinery, including ATP synthase activity. Research in Arabidopsis has shown that chloroplast gene expression responds to light-induced changes in the redox state of electron carriers . Similar regulatory mechanisms might affect ATP synthase subunit c function or expression in rice, with implications for dynamic photosynthetic responses.

Drought stress significantly impacts photosynthetic performance in crops. Interestingly, research has shown strong overlap between drought stress and norflurazon-induced transcriptomic changes in Arabidopsis , suggesting connections between drought responses and chloroplast signaling. The ATP synthase, as a key component of energy metabolism, might be involved in these drought response pathways.