Recombinant Oryza sativa subsp. japonica Probable DNA gyrase subunit A, chloroplastic/mitochondrial (GYRA), partial

What is DNA gyrase subunit A in Oryza sativa and what is its fundamental role?

DNA gyrase subunit A (GYRA) in Oryza sativa is a critical component of the DNA gyrase enzyme complex present in both chloroplasts and mitochondria. GYRA works in conjunction with the GyrB subunit to introduce negative supercoils into DNA, which is essential for proper DNA replication, transcription, and recombination in these organelles. Unlike most eukaryotes that lack DNA gyrase, plants have retained this bacterial-like enzyme for organellar DNA management .

The protein contains domains for DNA breakage and reunion, which are essential for its topoisomerase activity. GYRA is encoded by nuclear genes but targeted to organelles where it performs its function in maintaining DNA topology.

Where is GYRA expressed and localized in rice cells?

GYRA in rice is dual-targeted to both chloroplasts and mitochondria. The protein contains targeting sequences that direct it to these organelles after synthesis in the cytoplasm. Expression studies have demonstrated that GYRA plays critical roles in both organelles, as depletion of GYRA affects both chloroplasts and mitochondria by reducing chloroplast numbers and inducing morphological and physiological abnormalities .

This dual targeting is consistent with the role of DNA gyrase in maintaining DNA topology in both organelles, which have distinct but related needs for DNA supercoiling regulation.

How does rice GYRA structurally compare to bacterial DNA gyrase A?

While both rice and bacterial GYRA share a similar core structure and function, there are notable differences:

The rice GYRA-GyrB interaction appears particularly strong, with evidence showing "the two subunits coeluted in stoichiometric amounts all through the urea wash," suggesting that "the interactions between the two subunits are stronger than those found for E. coli DNA gyrase" .

What specific mutations in GYRA affect its function in DNA supercoiling?

Several key amino acid substitutions have been identified that affect gyrase function, particularly in the context of quinolone resistance. Though primarily studied in bacterial systems, these mutations occur in the highly conserved Quinolone Resistance-Determining Region (QRDR):

What is the role of GYRA in chloroplast genome maintenance and replication?

GYRA plays a critical role in chloroplast genome maintenance and replication. Research has demonstrated that depletion of GYRA affects chloroplasts by "reducing chloroplast numbers and inducing morphological and physiological abnormalities" .

Flow cytometry analysis revealed that "the average DNA content in the affected chloroplasts was significantly higher than in the control organelles," suggesting defects in DNA replication or partitioning. Abnormal chloroplasts contained "one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma," indicating that GYRA is essential for proper nucleoid organization and division .

These findings suggest that GYRA is necessary for:

• Proper chloroplast DNA replication

• Nucleoid organization and partitioning

• Maintenance of DNA topology essential for gene expression

• Division and segregation of nucleoids during chloroplast division

How does GYRA contribute to nucleoid partitioning in rice chloroplasts?

GYRA plays a "critical role in chloroplast nucleoid partitioning by regulating DNA topology" . Mechanistically, GYRA contributes to nucleoid partitioning through:

• Introducing negative supercoils into chloroplast DNA, affecting its compaction and organization

• Resolving topological issues that arise during DNA replication and transcription

• Facilitating proper segregation of newly replicated DNA into daughter nucleoids

• Maintaining appropriate DNA topology needed for nucleoid division

When GYRA function is compromised, pulse-field gel electrophoresis reveals that "the sizes and/or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant" . This results in abnormal chloroplasts containing "one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma," highlighting GYRA's essential role in maintaining proper nucleoid structure and organization.

What are the evolutionary implications of GYRA conservation between rice and other plant species?

The conservation of GYRA across plant species suggests it plays an essential role in plant organellar biology that has been maintained through evolution. The presence of DNA gyrase in plant organelles, but not in the nuclear genome or in most other eukaryotes, points to the bacterial origin of chloroplasts and mitochondria.

Comparative genomics between rice (Oryza sativa) and Arabidopsis thaliana shows these distantly related flowering plants "share similar sets of known functional domains, although there may be several functionally important domains unique to each lineage" . This suggests that while the core functions of GYRA are conserved, species-specific adaptations may exist.

The evolutionary patterns seen in rice genes more broadly indicate that "natural selection may have played a role for duplicated genes in both species, so that duplication was suppressed or favored in a manner that depended on the function of a gene" . This evolutionary pressure likely applies to essential genes like GYRA as well.

What are the optimal conditions for expressing and purifying recombinant rice GYRA?

Based on available research, the following protocols are recommended for recombinant rice GYRA:

For purification, a multi-step approach is recommended:

• Initial capture using affinity chromatography (His-tag or GST-tag)

• Secondary purification via ion exchange chromatography

• Final polishing using size exclusion chromatography

Specific buffer conditions identified for stable GYRA activity include:

• 50 mM Tris-HCl (pH 7.5-8.0)

• 50-100 mM KCl or potassium glutamate

• 5-10 mM MgCl₂

• 1-2 mM ATP (for activity assays)

• 1-5 mM DTT

• 10% glycerol for storage

Research indicates that rice GYRA shows unusual stability in urea, as "some GyrA and GyrB polypeptides were still coeluted with gyrase activity after washing with 8 column volumes of 5 M urea" .

What techniques are most effective for visualizing GYRA localization in rice organelles?

Several complementary techniques can be used to visualize GYRA localization in rice organelles:

For co-localization studies, DAPI staining can be used to visualize nucleoids, which revealed in previous research that abnormal chloroplasts "contained one or a few large nucleoids instead of multiple small nucleoids" when GYRA function was compromised.

How can researchers effectively generate knockout or knockdown models of GYRA in rice?

Based on current methodologies, several approaches can be used to manipulate GYRA expression:

When designing these experiments, it's important to note that complete loss of GYRA function may be lethal, given its essential role in organellar DNA maintenance. Previous research observed that "depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles" .

The rice genome annotation project identified "almost 5000 annotated protein-coding genes were found to be disrupted in insertional mutant lines" , which could potentially include GYRA mutants that might be available in existing collections.

How can researchers distinguish between the chloroplastic and mitochondrial functions of GYRA?

Since GYRA is dual-targeted to both chloroplasts and mitochondria, distinguishing its functions in each organelle requires specialized approaches:

• Organelle-specific targeting:

  • Generate modified GYRA constructs with enhanced specificity for either chloroplasts or mitochondria

  • Express these constructs in GYRA-depleted backgrounds to assess organelle-specific rescue

• Differential phenotyping:

  • Chloroplast-specific assays: Photosynthetic efficiency, chloroplast nucleoid organization

  • Mitochondria-specific assays: Respiration rates, mitochondrial DNA content

• Organelle isolation techniques:

  • Density gradient centrifugation to separate pure chloroplast and mitochondrial fractions

  • Analyze GYRA activity and DNA topology in each isolated organelle type

• Organelle-specific inhibitors:

  • Apply inhibitors that preferentially affect one organelle type

  • Analyze differential responses to distinguish organelle-specific GYRA functions

Flow cytometry analysis can be particularly valuable, as it previously revealed that "the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles" .

What are the best approaches for studying GYRA-protein interactions in rice?

Multiple complementary techniques can be employed to identify and characterize GYRA-protein interactions:

The natural interaction between GYRA and GyrB serves as a positive control for these studies, as these subunits are known to interact to form the functional DNA gyrase holoenzyme. Research has shown this interaction is particularly strong in rice, as "the two subunits coeluted in stoichiometric amounts all through the urea wash" .

What correlations exist between GYRA function and agronomic traits in rice?

While direct correlations between GYRA and agronomic traits aren't explicitly detailed in the search results, we can infer potential relationships based on the essential role of GYRA in chloroplast and mitochondrial function.

Research on rice genetics has identified numerous genes associated with important agronomic traits:

• Yield-related genes include CL4 and d35

• Plant height genes include OsIRX10, HDT702, and sd1

• Flowering time is regulated by genes like dh1

Since GYRA is essential for proper organelle function, and these organelles are critical for plant energy metabolism, mutations or variations in GYRA efficiency could potentially impact:

Future research could explore whether natural variation in GYRA sequences among rice varieties correlates with differences in agronomic performance, particularly under stress conditions.