Recombinant Nicotiana tabacum Cytochrome b-c1 complex subunit Rieske-5, mitochondrial

What is the Rieske FeS protein and what is its role in Nicotiana tabacum?

The Rieske FeS protein is a core subunit of the cytochrome b6f complex in chloroplasts and the cytochrome b-c1 complex in mitochondria. In tobacco (Nicotiana tabacum), the Rieske FeS protein plays a crucial role in electron transport chains. In chloroplasts, it is part of the cytochrome b6f complex that catalyzes a rate-limiting step in thylakoid electron transport, representing an important regulatory point for photosynthesis . In mitochondria, the Rieske protein (particularly Rieske-2) functions as part of the cytochrome b-c1 complex (Complex III) in the respiratory electron transport chain, facilitating electron transfer and contributing to ATP production .

How does the Rieske protein contribute to electron transport in tobacco plants?

The Rieske FeS protein contains an iron-sulfur cluster that facilitates electron transfer from the plastoquinone pool to cytochrome f in chloroplasts or from ubiquinol to cytochrome c1 in mitochondria. In tobacco, studies have shown that the Rieske protein is critical for maintaining efficient electron flow through the cytochrome b6f complex. When overexpressed, it can increase the abundance of functional cytochrome b6f by up to 40%, enhancing cytochrome f activity . This increased activity leads to a more oxidized primary quinone acceptor of Photosystem II (QA) and plastoquinone pool, enabling faster electron transport from the plastoquinone pool to Photosystem I upon changes in irradiance .

What expression systems are optimal for producing recombinant tobacco Rieske proteins?

Recombinant tobacco Rieske proteins can be effectively produced using E. coli expression systems. Based on available data, E. coli has been successfully used to express the full-length Nicotiana tabacum cytochrome b-c1 complex subunit Rieske-2 protein (residues 61-272) . For optimal expression:

• Use a vector with an appropriate promoter (T7 is common for high-level expression)

• Include a His-tag (typically N-terminal) to facilitate purification

• Express the mature protein sequence (without transit peptide)

• Optimize codon usage for E. coli if necessary

• Consider expressing at lower temperatures (16-20°C) to enhance proper folding

Purification can be achieved using metal affinity chromatography (for His-tagged proteins), followed by additional chromatographic steps if higher purity is required .

How should recombinant Rieske proteins be stored and handled for optimal stability?

For optimal stability and activity of recombinant tobacco Rieske proteins:

Before opening, centrifuge vials briefly to bring contents to the bottom. After reconstitution, aliquot the protein to avoid repeated freeze-thaw cycles which can cause denaturation and loss of activity .

What analytical techniques are recommended for assessing the purity and functionality of recombinant Rieske proteins?

Several analytical techniques can be employed to assess recombinant Rieske protein quality:

• Purity Assessment:

  • SDS-PAGE (>90% purity standard)

  • Western blotting with anti-Rieske or anti-His antibodies

  • Mass spectrometry for accurate molecular weight determination

• Functionality Assessment:

  • Cytochrome f activity assays (in vitro)

  • Electron paramagnetic resonance (EPR) spectroscopy to assess iron-sulfur cluster integrity

  • Spectrophotometric assays measuring electron transfer rates

  • Circular dichroism to evaluate secondary structure integrity

• Complex Formation Assessment:

  • Blue native PAGE to evaluate incorporation into cytochrome complexes

  • Co-immunoprecipitation with other complex components

When evaluating cytochrome f activity to confirm Rieske protein functionality, researchers have observed up to 20% increases in functional cytochrome b6f complex activity in tobacco plants with Rieske overexpression .

How does overexpression of Rieske FeS protein affect photosynthetic parameters in tobacco?

Overexpression of Rieske FeS protein in tobacco leads to several significant changes in photosynthetic parameters:

• Structural Changes:

  • Up to 40% increase in cytochrome b6f complex abundance

  • Enhanced in vitro cytochrome f activity indicating full functionality of the complex

• Electron Transport Modifications:

  • More oxidized primary quinone acceptor of Photosystem II (QA)

  • More oxidized plastoquinone pool

  • Faster electron transport from plastoquinone pool to Photosystem I upon irradiance changes

• Non-Photochemical Quenching Effects:

  • Faster establishment of qE (energy-dependent component of non-photochemical quenching)

  • More rapid build-up of transthylakoid proton gradient

  • Enhanced regulation of cytochrome b6f activity

Interestingly, despite these improvements in electron transport components, studies have not consistently shown increases in steady-state rates of electron transport or CO2 assimilation in Rieske-overexpressing tobacco plants grown in either laboratory conditions or field trials. This suggests that the in vivo activity of the complex might only be transiently increased upon changes in irradiance, and that other factors may be limiting photosynthetic capacity in tobacco under tested conditions .

Why might Rieske overexpression effects differ between tobacco and other plant species?

The differential effects of Rieske overexpression between tobacco and other species (such as Arabidopsis thaliana and Setaria viridis) present an intriguing research question. Several hypotheses might explain these differences:

Research has shown that while Rieske overexpression led to increases in electron transport and CO2 assimilation rates in Arabidopsis thaliana and Setaria viridis, similar consistent increases were not observed in tobacco . This contradictory finding suggests that the cytochrome b6f complex may not be the only rate-limiting step in tobacco photosynthesis under tested CO2 and irradiance regimes .

What techniques can be used to study the impact of environmental toxicants on Rieske protein function in tobacco mitochondria?

Studying the impact of environmental toxicants (such as tobacco smoke components) on mitochondrial Rieske protein function requires sophisticated methodological approaches:

• Exposure Systems:

  • Controlled tobacco smoke exposure chambers for whole plants

  • Cell culture systems with tobacco smoke extract or isolated compounds

  • Quantifiable dosing of specific toxicants (acrolein, carbon monoxide, cyanide, etc.)

• Functional Assays:

  • Mitochondrial oxygen consumption measurements (respirometry)

  • Membrane potential assessment using fluorescent probes

  • ATP production quantification

  • Electron transport chain complex activity assays

• Molecular Analysis:

  • Evaluation of mtDNA damage and copy number

  • Analysis of Rieske protein oxidative modifications

  • Assessment of iron-sulfur cluster integrity

  • Protein expression and turnover rates

• Structural Analysis:

  • Changes in mitochondrial morphology (microscopy)

  • Assessment of mitophagy rates

  • Evaluation of supercomplexes via blue native PAGE

Research has shown that tobacco smoke components can significantly impair mitochondrial function through multiple mechanisms. Active and passive cigarette smoke decrease mitochondrial respiration and membrane potential, leading to decreased ATP content and increased oxidant production in various tissues in a dose- and time-dependent manner . Components of tobacco smoke, such as acrolein, can form bulky DNA adducts that may persist in mitochondrial DNA due to the inefficiency of mitochondrial DNA polymerase γ . Understanding these effects specifically on Rieske protein function could provide insights into mitochondrial dysfunction in tobacco-related diseases.

How do chloroplastic and mitochondrial Rieske proteins in tobacco differ in structure and function?

Tobacco plants contain both chloroplastic (cytochrome b6f complex) and mitochondrial (cytochrome b-c1 complex) Rieske proteins, which share core features but differ in several important aspects:

Both proteins contain characteristic [2Fe-2S] clusters coordinated by conserved cysteine and histidine residues, but their specific amino acid sequences and structural contexts differ to match their functional roles in their respective organelles. The chloroplastic Rieske is involved in photosynthetic electron transport and responds to light conditions, while the mitochondrial Rieske participates in respiratory electron transport and responds to metabolic demands .

What experimental design would best address contradictory findings regarding Rieske overexpression effects on photosynthesis?

To resolve contradictory findings regarding the effects of Rieske overexpression on photosynthesis in different species, a comprehensive experimental design would include:

• Multi-species Comparative Analysis:

  • Include tobacco, Arabidopsis, Setaria viridis, and other relevant species

  • Use identical transformation constructs and expression systems

  • Generate multiple independent transgenic lines with varying expression levels

• Comprehensive Phenotyping:

  • Measure Rieske protein levels and cytochrome complex abundance

  • Assess cytochrome f activity in standardized assays

  • Evaluate electron transport rates under multiple conditions

  • Measure CO2 assimilation under varying light and CO2 concentrations

  • Determine growth and yield parameters in controlled and field conditions

• Dynamic Responses Assessment:

  • Study transient responses to changing light conditions

  • Analyze induction and relaxation kinetics of photosynthesis

  • Measure non-photochemical quenching parameters

  • Evaluate plastoquinone redox state dynamics

• Limiting Factor Analysis:

  • Manipulate potential downstream limiting factors (Rubisco, ATP synthase)

  • Apply metabolic control analysis to determine flux control coefficients

  • Use mathematical modeling to identify species-specific bottlenecks

• Environmental Response Profiling:

  • Test responses under different temperature regimes

  • Evaluate drought and high light stress responses

  • Assess adaptation to fluctuating conditions

This comprehensive approach would help identify why overexpression of Rieske in Arabidopsis and Setaria led to increases in electron transport and CO2 assimilation rates, while similar consistent increases were not observed in tobacco despite increased cytochrome b6f complex abundance and activity .

How might combining Rieske overexpression with other genetic modifications enhance photosynthetic efficiency in tobacco?

Given that Rieske overexpression alone did not consistently enhance steady-state photosynthesis in tobacco despite increasing cytochrome b6f abundance, combining this modification with other genetic interventions might yield more substantial improvements:

• Co-manipulation of Multiple Electron Transport Components:

  • Simultaneous overexpression of plastocyanin or ferredoxin

  • Enhancing ATP synthase capacity to utilize the increased proton gradient

  • Optimizing cytochrome b6f subunit stoichiometry

• Integration with Carbon Fixation Enhancements:

  • Improving Rubisco catalytic efficiency or abundance

  • Enhancing regeneration of RuBP in the Calvin-Benson cycle

  • Engineering more efficient photorespiratory bypasses

• Coordination with Regulatory Mechanisms:

  • Modifying NPQ induction and relaxation kinetics

  • Enhancing state transitions for optimal light harvesting

  • Improving stomatal responses to maximize CO2 availability

• Stress Tolerance Integration:

  • Combining with heat stability enhancements for photosynthetic machinery

  • Incorporating drought tolerance mechanisms

  • Engineering improved recovery from photoinhibition

Research suggests that overexpression of Rieske FeS in tobacco may have the potential to increase plant productivity if combined with other traits . This indicates that while Rieske overexpression provides up to 20% increase in functional cytochrome b6f complex, other factors become limiting in tobacco that would need to be addressed concurrently to achieve significant productivity gains .

What role might mitochondrial Rieske proteins play in tobacco's response to environmental stresses?

Mitochondrial Rieske proteins likely play significant roles in tobacco's response to environmental stresses through several mechanisms:

• Oxidative Stress Management:

  • Maintaining electron flow through the respiratory chain during stress

  • Preventing excessive ROS formation at Complex III

  • Contributing to cellular redox balance maintenance

• Energy Production During Stress:

  • Sustaining ATP production under adverse conditions

  • Supporting energy-dependent stress response mechanisms

  • Enabling metabolic adjustments to changing environments

• Retrograde Signaling:

  • Participating in mitochondrial status communication to the nucleus

  • Contributing to stress-responsive gene expression programs

  • Influencing whole-plant acclimation responses

• Interaction with Environmental Toxicants:

  • Serving as targets for tobacco smoke components and air pollutants

  • Modulating respiratory adjustments to xenobiotic exposure

  • Influencing mitochondrial damage and repair mechanisms

Research has shown that mitochondria are highly sensitive to environmental toxicants and individual components of tobacco smoke and air pollution . Environmental toxicant exposure induces changes in mitochondrial respiration and metabolism, which could involve alterations in Rieske protein function. Components found in tobacco smoke can decrease mitochondrial respiration and membrane potential, impair proton pumping efficiency, and lead to decreased ATP production . Understanding how mitochondrial Rieske proteins specifically respond to these stressors could provide insights into stress adaptation mechanisms in tobacco plants.