Recombinant Oryza sativa subsp. japonica Probable nucleoredoxin 1-2 (Os03g0405900, LOC_Os03g29240), partial

What is the basic structure of rice nucleoredoxin 1-2?

Rice nucleoredoxin 1-2 (Os03g0405900, LOC_Os03g29240) belongs to the thioredoxin (TRX) superfamily. Based on structural analysis of plant nucleoredoxins, it likely contains multiple TRX-like domains with conserved redox-active sites. Plant nucleoredoxins are classified into three types based on domain structure: Type I contains three TRX-like domains where the first and third domains contain typical WCG/PPC redox active sites; Type II contains two TRX-like domains with atypical active sites; and Type III contains two TRX-like domains with highly conserved WCRPC and WCPPC/F/S active sites . To determine the specific type of Os03g0405900, researchers should perform sequence alignment with characterized nucleoredoxins and identify the number and nature of its redox-active domains.

How does rice nucleoredoxin 1-2 differ from other plant nucleoredoxins?

While specific information about Os03g0405900 differences is limited in the provided search results, plant nucleoredoxins show variability in their domain organization and active site compositions. For comparison, maize nucleoredoxin contains three TRX-like domains with the first and third domains containing the WCPPC active site of typical TRX-like domains . GbNRX1 from cotton (Gossypium barbadense) contains active sites WCG/CPC in its first and third domains . The Arabidopsis genome encodes both Type I and Type II nucleoredoxins (AtNRX1 and AtNRX2) . Rice nucleoredoxin 1-2 should be analyzed for its unique features through comparative sequence analysis, evolutionary studies, and biochemical characterization of its redox activities.

What are the primary biochemical functions of nucleoredoxin 1-2 in rice?

Based on studies of nucleoredoxins across plant species, rice nucleoredoxin 1-2 likely functions in disulfide bond reduction and regulation of redox homeostasis. Nucleoredoxins generally exhibit disulfide bond reduction ability in vitro, as demonstrated with maize NRX, GbNRX1, and both AtNRX1 and AtNRX2 . Additionally, proteomics data from wild rice (Oryza rufipogon) shows that nucleoredoxin 1-2 is significantly upregulated (2.22-fold) under phosphorus deficiency stress . This suggests a potential role in phosphorus stress response pathways, possibly through redox regulation of target proteins involved in phosphorus acquisition or metabolism.

What transcription factors regulate nucleoredoxin 1-2 expression?

The provided search results do not contain specific information about transcription factors regulating Os03g0405900. To identify potential regulatory elements, researchers should perform in silico analysis of the promoter region to identify putative transcription factor binding sites, especially those related to stress responses and phosphate starvation response elements. Chromatin immunoprecipitation (ChIP) assays and yeast one-hybrid screens could then be employed to confirm interactions between candidate transcription factors and the nucleoredoxin 1-2 promoter. Electrophoretic mobility shift assays (EMSA) would further validate these interactions in vitro.

How does nucleoredoxin 1-2 contribute to phosphorus deficiency tolerance in rice?

Proteomics analysis identified nucleoredoxin 1-2 (LOC_Os03g29240) as significantly upregulated (2.22-fold increase, p=0.0006) under low phosphorus conditions in wild rice . This suggests it plays a role in phosphorus deficiency response mechanisms. While the exact function has not been fully characterized, nucleoredoxins generally regulate protein activity through reduction of disulfide bonds. In this context, nucleoredoxin 1-2 may modify the activity of proteins involved in phosphorus acquisition, mobilization, or use efficiency. To investigate this further, researchers should identify interaction partners under phosphorus deficiency using co-immunoprecipitation followed by mass spectrometry, and characterize the impact of these interactions on phosphorus homeostasis through enzyme activity assays and metabolic profiling.

What is the relationship between nucleoredoxin 1-2 and reactive oxygen species (ROS) management during stress?

While specific information about Os03g0405900 and ROS is not provided in the search results, nucleoredoxins in other plants have demonstrated roles in ROS regulation. For example, AtNRX1 in Arabidopsis is involved in regulating ROS levels in seedlings, and NRX1 targets enzymes in hydrogen peroxide clearance pathways, including catalase (CAT) . NRX1 can regulate CAT activity, enhancing detoxification of H₂O₂ and protecting antioxidant enzymes from oxidative stress . To investigate whether rice nucleoredoxin 1-2 functions similarly, researchers should measure ROS levels and antioxidant enzyme activities in nucleoredoxin 1-2 overexpression and knockout/knockdown lines under various stress conditions. Biochemical assays could determine if rice nucleoredoxin 1-2 directly interacts with and regulates the activity of antioxidant enzymes.

How does nucleoredoxin 1-2 expression correlate with other stress-responsive genes?

The search results do not provide specific data on co-expression patterns of nucleoredoxin 1-2 with other genes. To address this question, researchers should conduct transcriptome and proteome analyses of wild-type and nucleoredoxin 1-2 transgenic plants under various stress conditions. This would identify genes whose expression patterns correlate with nucleoredoxin 1-2, potentially revealing functional networks. RNA-seq data analysis should focus on identifying co-expression modules and gene ontology enrichment to determine biological processes associated with nucleoredoxin 1-2 function. Validation of key correlations through qRT-PCR and protein abundance measurements would strengthen these findings.

What are the most effective methods for recombinant expression and purification of rice nucleoredoxin 1-2?

For successful recombinant expression and purification of rice nucleoredoxin 1-2, researchers should consider the following methodology:

• Expression system selection: E. coli BL21(DE3) is commonly used for expressing plant proteins, though eukaryotic systems like yeast may provide better folding for multi-domain proteins like nucleoredoxins.

• Vector design: Use a vector containing an N-terminal His-tag or GST-tag for purification, with a cleavable linker. Based on approaches used for other plant proteins, the gene should be codon-optimized for the expression host.

• Induction conditions: Optimize temperature (typically 16-25°C for multi-domain proteins), IPTG concentration (0.1-1.0 mM), and induction duration (4-16 hours) to maximize soluble protein production.

• Purification protocol:

  • Affinity chromatography using Ni-NTA for His-tagged proteins

  • Size exclusion chromatography to ensure protein homogeneity

  • Consider ion exchange chromatography as a polishing step

• Protein activity verification: Conduct insulin disulfide reduction assays to confirm that the purified protein retains its redox activity, similar to methods used for wheat TaNRX1 .

What assays can be used to measure the disulfide reduction activity of nucleoredoxin 1-2?

Several established assays can be used to measure the disulfide reduction activity of recombinant nucleoredoxin 1-2:

• Insulin disulfide reduction assay: This is the classical approach used for various nucleoredoxins, including those from wheat and cotton . The assay measures the ability of nucleoredoxin to reduce disulfide bonds in insulin, causing insulin chain precipitation that can be monitored by increased turbidity at 650 nm.

• DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)) reduction assay: This colorimetric assay measures the production of TNB (2-nitro-5-thiobenzoate) when the disulfide bond in DTNB is reduced, resulting in a yellow color that can be quantified at 412 nm.

• Fluorescent substrate assays: Using fluorescently labeled peptides containing disulfide bonds, where fluorescence increases upon reduction.

• Redox-sensitive GFP (roGFP) assay: Can be used to monitor nucleoredoxin activity in vivo by expressing roGFP fusion proteins and measuring fluorescence ratio changes that reflect redox state alterations.

For domain-specific analysis, researchers should express each TRX-like domain separately and compare their disulfide reduction activities, similar to the approach used for wheat TaNRX1 where all three domains showed insulin disulfide reduction activity, with the third domain exhibiting the strongest activity .

What are the most effective strategies for creating nucleoredoxin 1-2 knockout or overexpression rice lines?

For effective genetic manipulation of nucleoredoxin 1-2 in rice, researchers should consider these approaches:

• CRISPR/Cas9 gene editing for knockout lines:

  • Design multiple sgRNAs targeting conserved redox-active sites

  • Use rice-optimized Cas9 systems with established rice transformation protocols

  • Screen transformants using PCR-based genotyping and sequence verification

  • Validate knockouts through RT-PCR and western blotting

• Overexpression strategies:

  • Use strong constitutive promoters (e.g., maize ubiquitin or rice actin) for consistent expression

  • Alternatively, use stress-inducible promoters to study context-specific functions

  • Include epitope tags (FLAG, HA, or GFP) to facilitate protein detection and purification

  • Generate multiple independent transgenic lines with varying expression levels

• Tissue-specific or inducible expression systems:

  • For studying developmental roles, employ tissue-specific promoters

  • For temporal control, use chemical-inducible expression systems like the glucocorticoid receptor system

The methodology demonstrated for RRA3 (another rice nucleoredoxin) could serve as a model, where both overexpression and knockout lines were successfully generated to study its function in ratooning ability .

How can protein-protein interactions of nucleoredoxin 1-2 be effectively identified and characterized?

To comprehensively identify and characterize protein-protein interactions of rice nucleoredoxin 1-2:

• In planta interactome identification:

  • Co-immunoprecipitation (Co-IP) using epitope-tagged nucleoredoxin 1-2 followed by mass spectrometry

  • Proximity-dependent biotin identification (BioID) or TurboID approaches

  • Split-ubiquitin or split-luciferase complementation screening

• Validation of interactions:

  • Yeast two-hybrid (Y2H) assays for direct interaction verification

  • Bimolecular fluorescence complementation (BiFC) for in vivo interaction confirmation

  • Förster resonance energy transfer (FRET) for real-time interaction dynamics

  • In vitro pull-down assays using recombinant proteins

• Interaction characterization:

  • Determine domains responsible for interaction through truncation analysis

  • Identify critical residues through site-directed mutagenesis

  • Assess if interactions are redox-dependent by using reducing/oxidizing conditions

  • Evaluate interaction kinetics using surface plasmon resonance (SPR)

For example, this approach could help determine whether rice nucleoredoxin 1-2 interacts with histidine kinases similar to how RRA3 interacts with OHK4 (a cytokinin receptor) to inhibit its dimerization through disulfide bond reduction .

How does rice nucleoredoxin 1-2 compare functionally with nucleoredoxins in other plant species?

Based on the available search results, we can draw some comparative insights about nucleoredoxins across plant species:

To comprehensively investigate functional conservation and divergence, researchers should conduct complementation experiments where rice nucleoredoxin 1-2 is expressed in nucleoredoxin mutants of other species (e.g., Arabidopsis nrx1) to determine if it can rescue the mutant phenotype. Additionally, phylogenetic analysis combined with structural modeling would provide insights into evolutionary relationships and potential functional specialization.

What unique amino acid sequence features distinguish rice nucleoredoxin 1-2 from other nucleoredoxins?

The search results do not provide specific sequence information for rice nucleoredoxin 1-2. To address this question, researchers should:

• Perform comprehensive sequence alignment of nucleoredoxin 1-2 with other plant nucleoredoxins, focusing on:

  • Conservation of redox-active sites (typically WCXXC motifs)

  • Presence and arrangement of TRX-like domains

  • Unique insertions or deletions

  • Subcellular localization signals

• Analyze domain architecture to determine if rice nucleoredoxin 1-2 is Type I, II, or III according to the classification described for plant nucleoredoxins .

• Conduct evolutionary rate analysis to identify rapidly evolving regions that might indicate functional specialization.

• Use structural homology modeling to predict three-dimensional structure and compare with known TRX structures.

• Examine post-translational modification sites that might regulate activity or localization.

This comprehensive analysis would reveal distinguishing features that could explain any functional specialization of rice nucleoredoxin 1-2 compared to other plant nucleoredoxins.

How might nucleoredoxin 1-2 be involved in phosphorus use efficiency breeding strategies?

Given that nucleoredoxin 1-2 is significantly upregulated (2.22-fold, p=0.0006) under phosphorus deficiency in wild rice , it represents a potential target for improving phosphorus use efficiency in cultivated rice. Researchers could exploit this finding through:

• Marker-assisted selection: Develop molecular markers for favorable alleles of nucleoredoxin 1-2 that confer enhanced phosphorus use efficiency.

• Promoter analysis and haplotype mining: Similar to the approach used for RRA3 where variations in the promoter were associated with ratooning ability , researchers should explore nucleoredoxin 1-2 promoter variations across rice germplasm to identify superior haplotypes with optimal expression under phosphorus deficiency.

• Transgenic approaches: Develop transgenic rice with controlled expression of nucleoredoxin 1-2, potentially using phosphorus deficiency-inducible promoters for targeted expression only when needed.

• CRISPR/Cas9 base editing: Fine-tune nucleoredoxin 1-2 expression through precise modifications of its promoter or regulatory regions rather than altering the coding sequence.

• Identification of interaction networks: Map the protein-protein interaction network of nucleoredoxin 1-2 under phosphorus deficiency to identify additional breeding targets that could enhance phosphorus acquisition and utilization.

Validation of these approaches would require field trials under varying phosphorus availability conditions, measuring multiple agronomic traits and phosphorus content in plant tissues.

What role might nucleoredoxin 1-2 play in integrating multiple stress response pathways?

While specific information on rice nucleoredoxin 1-2's role in multiple stress integration is limited in the search results, research on nucleoredoxins in other plants suggests potential mechanisms:

• Redox signaling hub: Nucleoredoxins can modulate the activity of multiple target proteins through redox regulation, potentially serving as integration points for different stress signaling pathways. For example, wheat TaNRX1 positively regulates drought stress tolerance through ROS management , while rice nucleoredoxin 1-2 responds to phosphorus deficiency .

• Hormone signaling regulation: The finding that another rice nucleoredoxin (RRA3) regulates cytokinin signaling by inhibiting receptor dimerization suggests nucleoredoxin 1-2 might similarly regulate hormone receptors or signaling components that mediate multiple stress responses.

• Metabolic pathway coordination: Nucleoredoxins in other systems interact with metabolic enzymes like phosphofructokinase 1 (PFK1) to regulate the balance between glycolysis and pentose phosphate pathways . Nucleoredoxin 1-2 might similarly regulate metabolic shifts required for adaptation to multiple stresses.

To investigate this hypothesis, researchers should perform transcriptome and proteome profiling of nucleoredoxin 1-2 transgenic lines under combined stresses (e.g., drought + phosphorus deficiency) and identify signaling nodes that are differentially regulated compared to single stresses.

How can structural analysis of nucleoredoxin 1-2 inform the design of redox-based agricultural biotechnology?

Advanced structural analysis of nucleoredoxin 1-2 could guide innovative agricultural biotechnology applications through:

• Structure-based protein engineering:

  • Modify redox-active sites to alter substrate specificity or catalytic efficiency

  • Design chimeric proteins combining domains from different nucleoredoxins to create novel functions

  • Introduce regulatory switches for conditional activation under specific stresses

• Rational design of chemical modulators:

  • Develop small molecules that can enhance or inhibit nucleoredoxin activity based on structural binding pockets

  • Create redox-responsive agrochemicals that work in concert with endogenous nucleoredoxin systems

• Biosensor development:

  • Design nucleoredoxin-based biosensors for real-time monitoring of cellular redox status in crop plants

  • Create diagnostic tools for early detection of redox imbalance during stress conditions

• Target site identification:

  • Use structural data to predict and validate interaction interfaces with partner proteins

  • Identify critical residues that determine specificity toward different target proteins

• Directed evolution platforms:

  • Design high-throughput screening systems based on structural insights to evolve nucleoredoxins with enhanced properties for stress tolerance

To achieve these applications, researchers would need to determine the crystal or NMR structure of rice nucleoredoxin 1-2, characterize its redox potential and reaction kinetics, and map the structural changes that occur upon oxidation/reduction and target protein binding.

What are the most promising research directions for rice nucleoredoxin 1-2 in addressing agricultural challenges?

Based on the available information, several promising research directions emerge:

• Phosphorus use efficiency improvement: Given nucleoredoxin 1-2's upregulation under phosphorus deficiency , investigating its role in phosphorus acquisition and utilization pathways could lead to improved varieties for low-input farming systems.

• Multi-stress tolerance: Exploring the potential role of nucleoredoxin 1-2 in integrating responses to combined stresses (particularly nutritional deficiencies with abiotic stresses) could address increasingly complex field conditions due to climate change.

• Redox regulation of development: The finding that another rice nucleoredoxin (RRA3) affects ratooning ability through cytokinin signaling suggests nucleoredoxin 1-2 might regulate important developmental processes through similar mechanisms.

• Comparative nucleoredoxin biology: A systematic comparison of nucleoredoxin functions across crop species (rice, wheat, maize) could reveal conserved mechanisms and species-specific adaptations for redox regulation.

• Nucleoredoxin haplotype mining: Identifying superior natural variants of nucleoredoxin 1-2 from diverse rice germplasm, particularly from wild relatives adapted to marginal conditions, could provide valuable genetic resources for breeding.

Each of these directions offers potential for improving rice productivity under challenging environmental conditions by leveraging the fundamental redox regulatory functions of nucleoredoxin 1-2.

What technological advances would accelerate understanding of nucleoredoxin 1-2 function in planta?

Accelerating our understanding of nucleoredoxin 1-2 function would benefit from these technological advances:

• In vivo redox imaging:

  • Development of genetically encoded redox sensors with subcellular resolution

  • Real-time visualization of nucleoredoxin activity in living plant cells

• Temporal control systems:

  • Optogenetic control of nucleoredoxin activity to study immediate downstream effects

  • Chemical-inducible nucleoredoxin systems for precise temporal regulation

• Interactome mapping technologies:

  • Proximity-dependent labeling approaches optimized for plant systems

  • Single-cell proteomics to reveal cell-type specific interaction networks

• Cryo-EM and structural analysis:

  • High-resolution structures of nucleoredoxin-target protein complexes

  • Time-resolved structural studies to capture reaction intermediates

• Systems biology approaches:

  • Multi-omics integration frameworks to connect nucleoredoxin activity with global cellular responses

  • Machine learning algorithms to predict nucleoredoxin targets and functions from large datasets

• Field phenotyping technologies:

  • High-throughput phenotyping platforms to assess nucleoredoxin 1-2 transgenic lines under field conditions

  • Sensors for non-destructive measurement of redox status in crop plants

These technological advances would bridge the gap between molecular mechanisms and field-relevant phenotypes, accelerating the translation of nucleoredoxin research into agricultural improvements.