Recombinant Oryza sativa subsp. indica ATP synthase subunit c, chloroplastic (atpH)

What is the role of ATP synthase in rice energy metabolism?

ATP synthase is critical for energy production in rice cells, particularly under environmental stress conditions. The enzyme complex catalyzes ATP synthesis using the proton gradient established during photosynthesis in chloroplasts. Research has shown that ATPase activity significantly influences heat resistance and yield characteristics in rice varieties. For instance, increased ATPase activity has been correlated with improved energy status in heat-resistant rice cultivars like ZLY30, where ATPase activity was 10.3% and 16.0% higher under elevated temperatures (33°C and 36°C, respectively) compared to control conditions (26°C) . Methodologically, researchers should assess both ATP content and ATPase activity to comprehensively understand energy metabolism.

How does ATP synthase activity vary between indica and japonica rice subspecies?

Significant variation exists in ATP synthase activity between indica and japonica subspecies, reflecting their evolutionary divergence and adaptation to different environments. When studying these differences, researchers should implement parallel experimental designs examining both subspecies under identical conditions. Studies comparing cultivars like ZLY30 (characterized by heat resistance, high yield, and high quality) with more susceptible varieties like LLY35 show distinct patterns in ATP metabolism . For proper subspecies comparison, utilize pure genetic lines of Oryza sativa subsp. indica and japonica, and employ quantitative trait locus (QTL) mapping to identify genetic determinants underlying differences in ATP synthase activity.

How does environmental pH affect ATP synthase activity in rice?

Soil acidification significantly impacts rice ATP synthase function and energy metabolism. Low pH conditions (pH 3.5) have been shown to dramatically inhibit plasma membrane Ca²⁺-ATPase activity in rice roots compared to control conditions (pH 5.5) . Simultaneously, expression levels of PM H⁺-ATPase isoform 7 are up-regulated under H⁺ stress, suggesting differential regulation of ATP synthase components as part of adaptive responses . To investigate pH effects on chloroplastic ATP synthase, researchers should utilize hydroponic systems with precise pH control (ranging from pH 3.5 to 5.5) and monitor changes in enzyme activity, gene expression, and physiological responses over time.

What methods are recommended for quantifying ATP synthase activity in rice tissues?

For accurate quantification of ATP synthase activity in rice tissues, a multi-parameter approach is necessary:

• ATP content measurement using luminescence-based assays (sensitivity range: 10⁻¹² to 10⁻⁶ moles)

• ATPase activity assays measuring inorganic phosphate release

• PARP activity assessment to monitor energy utilization pathways

When comparing varieties, significant differences in ATP metabolism have been observed under stress conditions. For example, under high temperature (36°C), heat-resistant cultivar ZLY30 showed 67.2% lower ATP content compared to control conditions, while heat-susceptible LLY35 exhibited 130.2% higher ATP content . These contrasting responses highlight the importance of measuring both ATP content and ATPase activity to understand energy utilization efficiency.

What is the subcellular localization pattern of ATP synthase subunit c in rice cells?

The chloroplastic ATP synthase subunit c is primarily localized in thylakoid membranes, forming the membrane-embedded portion of the F₀ complex. To determine precise subcellular localization, implement:

• Cell fractionation techniques to isolate pure chloroplasts

• Immunogold electron microscopy using subunit c-specific antibodies

• Confocal microscopy with fluorescently-tagged recombinant proteins

Chloroplast isolation protocols should be optimized for rice tissue, as conventional protocols developed for Arabidopsis may yield suboptimal results with rice samples. Maintain sample temperature below 4°C throughout isolation to preserve membrane integrity and prevent protein denaturation.

What expression systems are optimal for recombinant production of rice atpH?

Several expression systems have been evaluated for recombinant production of membrane proteins from rice, with each offering distinct advantages:

For functional studies of atpH, E. coli BL21(DE3) has been successfully used for heterologous expression of rice proteins, as demonstrated in studies of rice immunity factors . When using this system, BL21(DE3) should be transformed with codon-optimized constructs and cultured at lower temperatures (16-20°C) after induction to improve proper folding of membrane proteins.

What are the critical factors for successful purification of recombinant atpH?

Purification of membrane proteins like ATP synthase subunit c requires careful optimization:

• Solubilization: Use mild detergents (DDM, LMNG) at concentrations just above CMC to extract without denaturation

• Affinity chromatography: Implement His-tag or other affinity tags for initial capture

• Size-exclusion chromatography: For final purification and buffer exchange

Critical parameters include maintaining pH stability (typically pH 7.0-8.0) and including glycerol (10-15%) to stabilize the protein. Researchers successfully purifying rice proteins have employed systematic purification approaches, utilizing ammonium sulfate precipitation (40-80% saturation) followed by column chromatography techniques including anion-exchange and gel filtration . For highly hydrophobic membrane proteins like atpH, specific detergent screening is essential for maintaining structural integrity.

How can recombinant atpH be functionally validated after purification?

Functional validation of purified recombinant atpH should include multiple complementary approaches:

• Circular dichroism spectroscopy to confirm secondary structure

• Reconstitution into liposomes for proton transport assays

• Binding assays with other ATP synthase subunits

Additionally, ATP hydrolysis activity can be measured using colorimetric phosphate release assays or coupled enzyme assays. Researchers must distinguish between native-like oligomeric states versus monomeric or aggregated forms using analytical ultracentrifugation or native PAGE. Activity should be compared to controls using inhibitors like oligomycin or DCCD to confirm specificity.

What expression vector systems are recommended for recombinant atpH production?

For optimal recombinant atpH expression, vector selection depends on the host system and experimental goals:

When working with rice proteins, pCold TF vectors have been successfully employed for expression of recombinant proteins in E. coli . For plant expression, an improved Agrobacterium-mediated transformation system has been developed specifically for recalcitrant indica rice cultivars, which could be adapted for atpH expression .

What are effective strategies to overcome inclusion body formation during atpH expression?

Membrane proteins like atpH frequently form inclusion bodies during heterologous expression. Effective strategies include:

• Lower induction temperature (16-18°C) and reduced IPTG concentration (0.1-0.2 mM)

• Fusion partners (TF, MBP, SUMO) to enhance solubility

• Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ)

• Expression as split domains if possible

For refolding from inclusion bodies, use a gradual dialysis approach with decreasing concentrations of mild denaturants while introducing appropriate detergents. Success rates can be improved by implementing high-throughput screening of refolding conditions (pH, salt, additives) in 96-well format.

How does atpH contribute to rice response to abiotic stresses?

The ATP synthase complex plays a central role in energy metabolism during stress responses. Research has demonstrated that ATPase activity significantly influences rice responses to heat stress. Heat-resistant rice cultivars (like ZLY30) show increased ATPase activity under elevated temperatures, while susceptible varieties (like LLY35) exhibit decreased activity . This suggests that enhanced ATP utilization, rather than ATP accumulation, contributes to stress resistance.

To investigate atpH-specific contributions:

• Generate transgenic rice lines with modified atpH expression

• Monitor changes in ATPase activity, ATP content, and ROS production under stress conditions

• Assess photosynthetic efficiency using chlorophyll fluorescence measurements

Low pH stress studies also reveal important insights, as ATPase activity is significantly affected by acidic conditions, with PM Ca²⁺-ATPase activity dramatically inhibited in rice roots at pH 3.5 .

What analytical techniques can measure proton translocation activity of recombinant atpH?

Proton translocation is a critical function of ATP synthase subunit c. Several techniques can assess this activity:

• Fluorescent pH indicators (ACMA, pyranine) in proteoliposomes

• Patch-clamp electrophysiology for direct current measurements

• Isotope exchange assays using tritiated water

When establishing proteoliposome assays, control experiments should include known inhibitors (oligomycin, DCCD) and uncouplers (FCCP, valinomycin) to validate assay specificity. Reconstitution conditions require optimization of protein:lipid ratios (typically 1:100 to 1:1000) and lipid composition (including POPC, POPE, and cardiolipin).

How can researchers study interactions between atpH and other ATP synthase subunits?

Investigating subunit interactions within the ATP synthase complex requires multiple approaches:

• Co-immunoprecipitation with antibodies against specific subunits

• Yeast two-hybrid or split-ubiquitin assays for membrane protein interactions

• FRET/BRET analyses for in vivo interaction studies

• Crosslinking mass spectrometry to identify interaction interfaces

When analyzing results, researchers should consider that transient interactions may be difficult to capture, and the detergent environment can significantly impact observed interaction patterns. Reconstitution experiments combining purified subunits can provide complementary evidence for functional interactions.

What approaches can assess the impact of point mutations on atpH function?

Systematic mutation analysis of atpH can reveal structure-function relationships:

• Site-directed mutagenesis of conserved residues

• Alanine-scanning of transmembrane regions

• Introduction of mutations identified in stress-resistant rice varieties

For functional assessment:

• Measure proton conductance in reconstituted systems

• Analyze ATP synthesis/hydrolysis rates

• Assess oligomeric assembly using native PAGE or crosslinking

Complementation studies in atpH knockout/knockdown lines provide the most physiologically relevant assessment of mutant functionality. When designing mutations, focus on the conserved polar residues in the transmembrane domain that are essential for proton translocation.

How does atpH gene expression change under different environmental conditions?

Gene expression analysis of atpH should employ multiple techniques:

• qRT-PCR with properly validated reference genes

• RNA-seq for genome-wide expression patterns

• Nuclear run-on assays to distinguish transcriptional vs. post-transcriptional regulation

Research has shown that stress conditions significantly alter the expression of genes involved in energy metabolism. For example, low pH exposure for two weeks significantly down-regulated the transcript levels of several genes in rice roots, including those encoding antioxidant enzymes . This suggests that energy metabolism genes, including those in the ATP synthase complex, are likely responsive to environmental stressors.

How does ATP synthase activity correlate with rice yield and quality traits?

ATP synthase activity shows significant correlation with both yield and quality traits in rice. Research has demonstrated that ATPase activity mediates the balance among heat-resistance, high-yield, and high-quality traits in rice . The heat-resistant, high-yield, high-quality cultivar ZLY30 exhibits increased ATPase activity under elevated temperatures, suggesting that improved energy utilization efficiency contributes to these desirable traits.

To investigate this correlation:

• Compare ATP synthase activity across varieties with contrasting yield and quality traits

• Track changes in energy metabolism parameters throughout grain development

• Analyze co-segregation of ATP synthase activity with yield QTLs in mapping populations

The significant differences in ATP content and ATPase activity between cultivars with contrasting traits highlight the importance of energy metabolism in determining agronomic performance .

What CRISPR-Cas9 strategies are optimal for modifying atpH in rice?

CRISPR-Cas9 modification of atpH requires specialized approaches:

• Design of highly specific sgRNAs targeting atpH while avoiding off-target effects

• Selection of appropriate promoters for Cas9 expression (e.g., rice ubiquitin promoter)

• Implementation of tissue culture protocols optimized for indica rice transformation

For chloroplast-encoded genes like atpH, plastid transformation approaches may be more appropriate than nuclear CRISPR systems. Researchers should implement careful phenotypic analysis of edited lines, as chloroplast gene modifications can have pleiotropic effects on photosynthesis and energy metabolism.

How can proteomics approaches be used to study ATP synthase complexes in rice?

Advanced proteomics techniques provide powerful tools for studying ATP synthase complexes:

• Blue-native PAGE coupled with mass spectrometry to characterize intact complexes

• Crosslinking mass spectrometry to map subunit interfaces

• Quantitative proteomics to measure stoichiometric changes under stress conditions

• Post-translational modification analysis to identify regulatory phosphorylation/acetylation sites

Sample preparation is critical, with gentle solubilization using digitonin or amphipol preferred for maintaining complex integrity. Label-free quantification or TMT labeling can enable comparative studies across different rice varieties or stress conditions.

What biophysical techniques can reveal the structure-function relationship of recombinant atpH?

Several biophysical approaches provide insights into atpH structure and function:

• Cryo-electron microscopy of reconstituted ATP synthase complexes

• Solid-state NMR of isotope-labeled atpH in lipid environments

• Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

• Single-molecule FRET to monitor conformational changes during catalysis

These techniques require highly pure, homogeneous protein preparations. For solid-state NMR, ¹³C/¹⁵N-labeled protein can be produced by growing expression hosts in minimal media with labeled glucose and ammonium chloride as sole carbon and nitrogen sources.

How can researchers study the interplay between ATP synthase activity and reactive oxygen species in rice stress responses?

The relationship between ATP synthase function and ROS metabolism is complex and bidirectional:

• ATP synthase activity influences ROS production through electron transport chain regulation

• ROS can damage ATP synthase components, affecting function

• Energy status determines ROS detoxification capacity

Research protocols should include:

• Simultaneous measurement of ATP synthesis and ROS production

• Analysis of antioxidant enzyme activities (SOD, CAT, APX) alongside ATP synthase activity

• Assessment of lipid peroxidation as an indicator of oxidative damage

Studies in rice have shown that stress conditions like low pH significantly affect both ATP metabolism and ROS-related enzymes, with SOD and CAT activities decreasing by up to 48% under acidic conditions (pH 3.5) . This suggests coordination between energy metabolism and antioxidant systems during stress responses.

What are effective strategies to overcome low expression yields of recombinant atpH?

Membrane proteins like atpH often express poorly in heterologous systems. To improve yields:

• Optimize codon usage for the expression host

• Test multiple fusion tags (His, MBP, SUMO, TF)

• Screen expression conditions systematically (temperature, induction time, media composition)

• Consider cell-free expression systems for difficult constructs

Successful expression of recombinant rice proteins has been achieved using the pCold TF vector system in E. coli BL21(DE3) , which utilizes a cold-shock promoter and trigger factor fusion to improve folding. For particularly challenging constructs, testing multiple construct boundaries by truncating non-essential regions can identify more expressible variants.

How can researchers address protein aggregation issues during atpH purification?

Aggregation is a common challenge when working with hydrophobic membrane proteins like atpH:

• Screen a panel of detergents (DDM, LMNG, GDN) at different concentrations

• Include stabilizing additives (glycerol, sucrose, specific lipids)

• Maintain low protein concentration during initial solubilization

• Implement on-column detergent exchange during purification

Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can distinguish between proper oligomeric assemblies and non-specific aggregates. Consider amphipathic polymers (amphipols) or nanodiscs for stabilizing the purified protein in a membrane-like environment.

What controls are essential when measuring ATP synthase activity in rice tissue extracts?

Rigorous controls are critical for accurate measurement of ATP synthase activity:

• Specific inhibitor controls (oligomycin, venturicidin) to distinguish ATP synthase activity from other ATPases

• Heat-inactivated samples to establish baseline

• Purified enzyme standards for quantitative calibration

• Time-course measurements to ensure linear reaction rates

When comparing different rice varieties, standardize tissue collection and extraction procedures to minimize variation. Research comparing ATP content and ATPase activity in different rice cultivars has revealed significant variations that correlate with stress resistance traits .

How can conflicting data on ATP synthase activity under stress conditions be reconciled?

Researchers may encounter contradictory findings regarding ATP synthase responses to stress:

• Document precise experimental conditions (temperature, duration, tissue type, developmental stage)

• Consider variety-specific responses (indica vs. japonica, resistant vs. susceptible)

• Distinguish between acute and chronic stress responses

• Examine both transcriptional and post-translational regulation

Studies have shown that heat-resistant and heat-susceptible rice varieties exhibit opposite patterns in ATP content and ATPase activity under elevated temperatures . The heat-resistant cultivar ZLY30 showed decreased ATP content but increased ATPase activity, while the susceptible cultivar LLY35 showed increased ATP content but decreased ATPase activity. This highlights the importance of considering genotype-specific responses when interpreting conflicting data.

What strategies can address challenges in reproducing published results on rice ATP synthase?

Reproducibility challenges in ATP synthase research may stem from:

• Undocumented differences in genetic background of rice varieties

• Variations in growth conditions and stress application protocols

• Differences in extraction methods affecting enzyme activity

• Use of different activity assay formulations

To improve reproducibility:

• Use well-characterized seed stocks from established repositories

• Document growth conditions comprehensively (light, temperature, humidity, soil composition)

• Standardize tissue sampling (developmental stage, time of day)

• Include multiple biological and technical replicates

When comparing results across studies, consider that genetic differences even within the same subspecies (indica) can lead to substantial phenotypic variation in stress responses and energy metabolism.