Recombinant Saccharum hybrid NAD (P)H-quinone oxidoreductase subunit J, chloroplastic (ndhJ)

What is the functional role of ndhJ in the chloroplast NDH complex of Saccharum hybrids?

The ndhJ subunit functions as an integral component of the chloroplast NAD(P)H dehydrogenase (NDH) complex, which participates in photosystem I (PSI) cyclic and chlororespiratory electron transport pathways. Similar to other NDH subunits like NdhB, NdhD, and NdhF that have been better characterized, ndhJ likely contributes to preventing over-reduction of the stroma under certain environmental conditions, thereby alleviating oxidative stress . The NDH complex is primarily localized to the stroma thylakoids and interacts with PSI to form a supercomplex that becomes fully assembled approximately 48 hours after illumination during chloroplast development .

To investigate ndhJ function specifically, researchers should consider:

• Generating knockout mutants using CRISPR-Cas9 technology

• Comparing photosynthetic parameters between wildtype and mutant plants

• Measuring PSI cyclic electron transport rates under various light conditions

• Analyzing stromal redox state changes during environmental stress

How does the structure of the ndhJ subunit compare to other NDH subunits in Saccharum species?

While the search results don't provide specific structural information about ndhJ, comparative analysis methods used for other NDH subunits can be applied. Researchers investigating this question should:

• Perform sequence alignment of ndhJ across different Saccharum varieties and related species

• Use homology modeling based on known structures of related NDH subunits

• Apply protein structure prediction tools, comparing results with the more well-characterized subunits like NdhB, NdhL and NdhM

• Examine conservation patterns between ndhJ and equivalent subunits in cyanobacteria

The analysis approach should follow methods similar to those used for studying other subunits, as illustrated in the comparison of mitochondrial genes across seven species that established the relationship between sugarcane and Sorghum .

What evidence exists for genetic variation of ndhJ among different Saccharum hybrids?

Based on patterns observed in other chloroplast and mitochondrial genes, researchers should approach this question by:

• Sequencing the ndhJ gene from multiple commercial hybrids and ancestral species

• Analyzing SNPs and small indels across different cultivars

• Creating a variant frequency table similar to the mitochondrial variant analysis shown below:

This approach enables identification of both common variants (likely present in ancestral species) and rare variants (potentially arising after hybridization events), providing insight into the evolutionary history of ndhJ in modern commercial sugarcane varieties .

What are the recommended approaches for recombinant expression of Saccharum hybrid ndhJ protein?

For successful recombinant expression of ndhJ from Saccharum hybrids, researchers should consider:

• Expression system selection: E. coli systems may be suitable for initial studies, but plant-based or insect cell systems might better preserve functional properties due to appropriate post-translational modifications.

• Codon optimization: Adjust the coding sequence based on the expression host's codon usage bias.

• Purification strategy:

  • Include appropriate affinity tags (His, GST, etc.)

  • Develop a multi-step purification protocol incorporating ion exchange chromatography

  • Consider native purification conditions to maintain protein-protein interactions

• Validation methods:

  • Western blotting using antibodies similar to those developed for NdhB

  • Mass spectrometry analysis

  • Activity assays measuring NAD(P)H-dependent plastoquinone reduction

Researchers should note that membrane proteins like ndhJ can be challenging to express in soluble form. Strategies such as fusion with solubility-enhancing partners or the use of appropriate detergents during purification may improve yields.

How can researchers investigate the interaction between ndhJ and other subunits of the NDH-PSI supercomplex?

Based on methodologies used for studying other NDH subunits, researchers investigating ndhJ interactions should:

• Apply blue native PAGE (BN-PAGE) followed by two-dimensional SDS-PAGE to separate the intact NDH-PSI supercomplex and identify its components, as demonstrated in studies of other NDH subunits .

• Perform co-immunoprecipitation experiments using antibodies against ndhJ and potential interacting partners.

• Utilize sucrose density gradient centrifugation to isolate intact supercomplexes from thylakoid membranes for further analysis.

• Implement time-course experiments during chloroplast development to track the incorporation of ndhJ into the NDH complex and subsequent interaction with PSI, similar to studies showing that the NDH complex exists as a monomer in etioplasts but forms the NDH-PSI supercomplex within 48 hours during chloroplast development .

• Create transgenic lines with tagged versions of ndhJ to facilitate pull-down assays and interaction studies.

The experimental approach should consider that NDH subunits may function differently depending on developmental stage and environmental conditions.

What are the most effective methods for analyzing ndhJ function in vivo within Saccharum hybrids?

To effectively analyze ndhJ function in vivo, researchers should implement a multi-faceted approach:

• Generate ndhJ knockout or knockdown lines using:

  • CRISPR-Cas9 genome editing

  • RNAi suppression

  • Antisense technology

• Phenotype characterization under various conditions:

  • Standard growth conditions

  • Varying light intensities

  • Drought stress

  • Temperature stress

  • Combined stressors

• Physiological measurements:

  • Chlorophyll fluorescence analysis to assess PSI and PSII performance

  • P700 absorbance measurements to evaluate PSI oxidation-reduction kinetics

  • Gas exchange parameters

  • Reactive oxygen species (ROS) accumulation

• Molecular analysis:

  • Transcriptome profiling to identify compensatory responses

  • Proteomics to detect changes in NDH complex composition

  • Metabolomics to assess downstream effects on carbon metabolism

• Complementation studies:

  • Reintroduction of native ndhJ

  • Introduction of ndhJ variants

  • Cross-species complementation with ndhJ from related species

These approaches will provide comprehensive insights into ndhJ function while accounting for potential compensatory mechanisms that might mask phenotypes in single-gene knockout studies.

What techniques can researchers use to study the assembly of ndhJ into the NDH complex during chloroplast development?

To study ndhJ assembly into the NDH complex during chloroplast development, researchers should consider the following methodological approach:

• Time-course experiments:

  • Grow plants in darkness to develop etioplasts

  • Expose to light and collect samples at regular intervals (0h, 6h, 12h, 24h, 48h)

  • Isolate thylakoid membranes at each time point

• Protein complex separation:

  • Solubilize membranes with appropriate detergents (e.g., dodecyl maltoside)

  • Perform BN-PAGE to separate intact complexes

  • Follow with two-dimensional SDS-PAGE to identify individual subunits

• Immunodetection:

  • Use antibodies against ndhJ and other NDH components

  • Track the transition of ndhJ from free protein to incorporation in the NDH monomer and subsequently the NDH-PSI supercomplex

• Quantitative analysis:

  • Measure protein abundance at each stage

  • Calculate assembly rates and efficiency

  • Compare with other NDH subunits

This approach parallels successful studies of other NDH subunits that demonstrated the NDH complex exists as a 550-kDa monomer in etioplasts but interacts with PSI to form a supercomplex within 48 hours during light-induced chloroplast development .

How can researchers differentiate between the roles of ndhJ in the NDH-PSI supercomplex versus its function as part of the NDH monomer?

Differentiating between ndhJ functions in different complex states requires sophisticated biochemical and genetic approaches:

• Complex separation and functional assessment:

  • Isolate NDH monomers and NDH-PSI supercomplexes using sucrose gradient centrifugation

  • Measure NAD(P)H dehydrogenase activity in both complexes

  • Assess electron transport capabilities under various substrate conditions

• Mutation analysis:

  • Design mutations in ndhJ that specifically disrupt interaction with PSI without affecting incorporation into the NDH monomer

  • Evaluate phenotypic consequences of these targeted disruptions

• Temporal expression manipulation:

  • Create inducible expression systems for modified ndhJ variants

  • Introduce these variants at different stages of chloroplast development

  • Measure impact on complex assembly and function

• Structural biology approaches:

  • Perform cryo-electron microscopy of isolated complexes

  • Map the position of ndhJ in both the monomer and supercomplex

  • Identify structural changes associated with complex formation

This approach will help determine whether ndhJ serves different roles depending on complex state, similar to observations with other NDH subunits that show developmental and functional transitions .

What are the best approaches for comparative analysis of ndhJ across different Saccharum species and related genera?

For comprehensive comparative analysis of ndhJ across Saccharum species and related genera, researchers should implement:

• Phylogenetic analysis:

  • Sequence ndhJ from multiple Saccharum species, including S. officinarum, S. spontaneum, and commercial hybrids

  • Include sequences from related genera such as Sorghum, which has been identified as closely related to sugarcane based on mitochondrial gene analysis

  • Construct phylogenetic trees to visualize evolutionary relationships

• Structural variation analysis:

  • Identify indels, SNPs, and other variations

  • Map these variations onto predicted protein structures

  • Create a structural variant table similar to the one used for mitochondrial genome comparisons:

• Expression analysis:

  • Compare transcript and protein abundance across species

  • Evaluate response to environmental stressors

  • Assess developmental regulation patterns

• Functional complementation:

  • Express ndhJ from different species in a model system lacking the native gene

  • Measure functional recovery to assess conservation of function

  • Identify species-specific differences in protein activity

This comprehensive approach will provide insights into the evolution of ndhJ and its conserved and divergent features across the Saccharum genus and related species.

What strategies can overcome the challenges of low protein yield when expressing recombinant ndhJ?

Low protein yield is a common challenge when expressing membrane-associated proteins like ndhJ. Researchers can implement these strategies:

• Expression system optimization:

  • Test multiple expression hosts (E. coli, yeast, insect cells)

  • Evaluate different cell lines within each host system

  • Optimize growth conditions (temperature, media composition, induction parameters)

• Protein engineering approaches:

  • Remove hydrophobic domains that may interfere with expression

  • Create fusion constructs with solubility-enhancing partners (MBP, SUMO, etc.)

  • Design truncated versions that retain functional domains

• Purification method refinement:

  • Develop specialized extraction protocols for membrane-associated proteins

  • Test various detergent types and concentrations

  • Implement on-column refolding techniques

• Co-expression strategies:

  • Co-express with chaperone proteins

  • Include other NDH subunits that might stabilize ndhJ

  • Add specific cofactors during expression

These approaches should be systematically tested and optimized for the specific properties of Saccharum hybrid ndhJ.

How can researchers address the challenge of distinguishing between ndhJ from different genomic origins in polyploid Saccharum hybrids?

Modern commercial sugarcane cultivars are complex polyploid hybrids derived primarily from S. officinarum with contributions from other Saccharum species . To distinguish ndhJ variants from different genomic origins:

• Haplotype-specific amplification:

  • Design primers targeting SNPs specific to each ancestral species

  • Implement allele-specific PCR techniques

  • Validate using known control samples from parental species

• Next-generation sequencing approaches:

  • Perform deep sequencing of the ndhJ locus

  • Apply bioinformatic tools to separate reads based on variant patterns

  • Reconstruct haplotype-specific sequences

• RNA-based analysis:

  • Conduct RNA-seq to identify which variants are expressed

  • Perform allele-specific RT-qPCR

  • Analyze whether certain variants show condition-specific expression

• Protein-level distinction:

  • Use mass spectrometry to identify peptides unique to each variant

  • Develop variant-specific antibodies when possible

  • Analyze post-translational modifications that might differ between variants

This approach parallels methods used for analyzing mitochondrial genome variants in Saccharum hybrids, where researchers identified variant distribution patterns across different cultivars and species .

What are the emerging technologies that could advance our understanding of ndhJ function in Saccharum hybrids?

Several cutting-edge technologies show promise for advancing ndhJ research:

• Cryo-electron microscopy:

  • Determine high-resolution structures of the NDH complex with ndhJ in place

  • Visualize conformational changes during electron transport

  • Map interaction interfaces with other proteins

• Single-molecule techniques:

  • Track dynamics of complex assembly in real-time

  • Measure conformational changes during function

  • Quantify electron transfer rates at the single-molecule level

• Genome editing technologies:

  • CRISPR-Cas9 for precise modification of ndhJ in sugarcane

  • Base editing for introducing specific mutations without double-strand breaks

  • Prime editing for more complex sequence changes

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize ndhJ localization in chloroplasts

  • FRET-based approaches to study protein-protein interactions

  • Correlative light and electron microscopy for structural context

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Network modeling to understand ndhJ's role in broader photosynthetic processes

  • Machine learning for identifying patterns in complex datasets

These technologies, applied in combination, will provide unprecedented insights into ndhJ function and regulation in Saccharum hybrids.

How might climate change-related stresses affect the function and importance of ndhJ in Saccharum hybrids?

The NDH complex helps alleviate oxidative stress under certain conditions , suggesting ndhJ may play important roles in climate change adaptation:

• Research approaches to investigate this include:

  • Controlled environment studies simulating future climate scenarios

  • Field trials in gradient conditions representing predicted climate changes

  • Comparison of ndhJ sequence, expression, and function across Saccharum cultivars adapted to different environments

• Physiological measurements to assess:

  • NDH complex activity under elevated CO₂

  • Function during heat waves and temperature fluctuations

  • Performance under drought conditions

  • Response to combined stresses (heat + drought, UV + heat)

• Genetic engineering strategies:

  • Develop climate-resilient variants of ndhJ

  • Test overexpression effects on stress tolerance

  • Create conditional expression systems activated during stress

• Long-term adaptation studies:

  • Monitor natural variation in ndhJ across geographic regions

  • Track changes in allele frequency in response to changing climate

  • Develop predictive models for ndhJ evolution under climate change scenarios

This research direction has significant implications for developing climate-resilient sugarcane varieties to ensure sustainable production in changing environments.