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

What is NAD(P)H-quinone oxidoreductase subunit H (ndhH) and what is its role in Saccharum hybrids?

NAD(P)H-quinone oxidoreductase subunit H (ndhH) is a crucial component of the NAD(P)H dehydrogenase complex located in chloroplasts. In Saccharum hybrids, this enzyme catalyzes the reduction of quinones and other electron acceptors using NADPH or NADH as electron donors. NdhH is specifically a 49 kDa subunit that forms part of a larger complex involved in cyclic electron flow around photosystem I, which helps balance the ATP/NADPH ratio during photosynthesis . This is particularly important under stress conditions, as it enables the plant to adjust its energy production based on environmental demands.

Where is the ndhH gene located in the Saccharum hybrid genome?

The ndhH gene in Saccharum hybrids is located in the chloroplast genome, not the nuclear genome. More specifically, comparative genomic analyses have shown that "the cp boundary genes were essentially similar in Saccharum species, with both the ndhH and ndhF genes located on the JSA (SSC/IRa) and JSB (SSC/IRb)" . These boundaries refer to the junctions between the small single-copy (SSC) region and the inverted repeat regions (IRa and IRb) of the chloroplast genome.

The chloroplast genome of Saccharum species is typically around 141,000-142,000 base pairs in length, with the SSC region spanning approximately 12,500 base pairs. This genomic organization is consistent across various Saccharum species, including S. fulvum (141,151 bp) and S. narenga (141,218 bp) , as shown in the following table:

The conservation of ndhH's location across Saccharum species indicates its fundamental importance in chloroplast function and suggests strong evolutionary pressure to maintain its genomic context.

What experimental designs are most effective for studying the function of ndhH in Saccharum hybrids?

When investigating ndhH function in Saccharum hybrids, several experimental design approaches yield meaningful results depending on the specific research questions:

• Completely randomized designs: Treatments (such as different environmental conditions or genetic modifications affecting ndhH) are assigned randomly to experimental units. This approach is effective when studying how ndhH responds to controlled environmental variables .

• Randomized block designs: Experimental units are organized into blocks of similar subjects, with treatments randomly assigned within each block. This is particularly useful for accounting for variation between different Saccharum cultivars or growing conditions .

• Factorial designs: These allow for the simultaneous study of multiple factors affecting ndhH function. For example, a 2×2 factorial design could examine both light intensity and temperature effects on ndhH expression or activity .

• Time series experiments: These are valuable for studying dynamic changes in ndhH expression or activity during development or in response to environmental changes over time .

• Comparative transcriptomics: Long-read sequencing technology has proven effective for capturing full-length transcripts without assembly artifacts, which is particularly important given the complex genome of Saccharum hybrids .

When designing experiments to study ndhH function, it's crucial to include appropriate controls and consider the polyploid nature of Saccharum hybrids, which may complicate genetic analyses. Statistical methods should be carefully selected to account for the complex genetic background and potential environmental interactions.

How can heterodimer expression systems be used to study the subunit functions of NAD(P)H-quinone oxidoreductase in Saccharum hybrids?

Heterodimer expression systems provide a powerful approach to dissect the functional contributions of individual subunits within multi-subunit complexes like NAD(P)H-quinone oxidoreductase. This methodology can be adapted from approaches used with other species:

• Vector design and construction:

  • Create two expression vectors: one encoding the wild-type ndhH subunit tagged with polyhistidine and another encoding a mutant version

  • For the mutant version, target conserved residues similar to the His-194→Ala mutation, which has been shown to dramatically increase the Km for NADPH in other systems

• Co-expression strategy:

  • Transform both vectors into an appropriate expression system (E. coli, yeast, baculovirus, or mammalian cells)

  • Optimize expression conditions to ensure balanced production of both subunits

• Purification protocol:

  • Use nickel nitrilotriacetate column chromatography under non-denaturing conditions

  • Implement stepwise elution with imidazole to separate heterodimers (H194A/HNQOR) from homodimers

  • Confirm the composition of purified heterodimers using SDS-PAGE, non-denaturing PAGE, and immunoblot analysis

• Functional characterization:

  • Analyze enzyme kinetics using various electron acceptors (both two-electron acceptors like 2,6-dichloroindophenol and menadione, and four-electron acceptors like methyl red)

  • Determine Km and kcat values for both NADPH and NADH with each substrate

  • Compare kinetic parameters of the heterodimer with those of wild-type and mutant homodimers

This approach has revealed that in some NAD(P)H-quinone oxidoreductases, "the subunits function independently with two-electron acceptors, but dependently with a four-electron acceptor" . Similar studies with ndhH from Saccharum hybrids could provide insights into the functional organization of this complex and potentially reveal species-specific adaptations.

What are the challenges in purifying recombinant ndhH from Saccharum hybrids and how can they be overcome?

Purification of recombinant ndhH from Saccharum hybrids presents several specific challenges that require methodological solutions:

• Expression system selection challenges:

  • Plant proteins often fold incorrectly in bacterial systems

  • Solution: Use eukaryotic expression systems such as yeast, baculovirus, or mammalian cells for proper post-translational modifications

  • Recommendation: Compare expression levels and proper folding across multiple systems before scaling up

• Solubility issues:

  • As a chloroplastic membrane-associated protein, ndhH tends to form inclusion bodies

  • Solution: Express as fusion proteins with solubility-enhancing tags (MBP, SUMO, or thioredoxin)

  • Alternative approach: Include glycerol in extraction buffers (typically 10-15%) to improve stability

• Purification strategy complications:

  • Multiple versions of ndhH may exist due to Saccharum's polyploid genome

  • Solution: Use affinity chromatography with highly specific tags, followed by ion exchange chromatography to separate variants

  • Validation step: Confirm identity through mass spectrometry and Western blot using specific antibodies

• Stability during storage:

  • ndhH may degrade quickly after purification

  • Solution: "Store working aliquots at 4 degrees C for up to one week. Repeated freezing and thawing is not recommended"

  • Additional approach: Add protease inhibitors and test various buffer compositions for optimal storage

• Activity verification:

  • Determining if the purified protein retains native activity

  • Solution: Develop activity assays using various electron acceptors like 2,6-dichloroindophenol and menadione

  • Control validation: Compare activity with chloroplast extracts from Saccharum hybrids

When working specifically with Saccharum hybrid proteins, it's important to acknowledge the complex genome structure resulting from interspecific hybridization between S. officinarum and S. spontaneum , which may lead to multiple slightly different versions of the protein requiring careful characterization.

What structural features of NAD(P)H-quinone oxidoreductase contribute to its function in Saccharum hybrids?

The structural features of NAD(P)H-quinone oxidoreductase are critical to understanding its function in Saccharum hybrids. Based on structural studies of similar enzymes, several key features can be identified:

Understanding these structural features provides insights into how NAD(P)H-quinone oxidoreductase functions in Saccharum hybrids and may reveal adaptations specific to these plants that contribute to their unique physiological properties.

What does comparative transcriptome analysis reveal about the expression of ndhH in Saccharum hybrids versus their progenitor species?

Comparative transcriptome analysis provides valuable insights into gene expression patterns in Saccharum hybrids compared to their progenitor species. For ndhH and related genes, this approach has revealed:

• Expression pattern inheritance:

  • Long read transcriptome sequencing of sugarcane hybrids and their progenitor species shows distinct patterns of gene inheritance

  • "Sugar related transcripts originated from S. officinarum while several stress and senescence related transcripts were from S. spontaneum in the hybrid"

  • For chloroplast genes like ndhH, expression patterns often reflect the maternal inheritance, typically from S. officinarum in commercial hybrids

• Novel isoform identification:

  • Transcriptome analysis has revealed "common isoforms among the three genotypes and unique isoforms specific to each genotype"

  • This suggests "high scope for improvement of the modern hybrids by utilizing novel gene isoforms from the progenitor species"

  • For ndhH, specific isoforms may contribute to differences in photosynthetic efficiency or stress tolerance

• Subgenomic contributions:

  • "In general, the hybrid shared a larger number of transcripts with S. officinarum than with S. spontaneum, reflecting the genomic contribution, while the progenitors shared very few transcripts between them"

  • This genomic bias influences the expression profile of genes involved in chloroplast function, including ndhH

• Functional enrichment patterns:

  • The hybrid demonstrated "a higher number of transcripts related to sugar transporters, invertases, transcription factors, trehalose, UDP sugars, and cellulose than the two progenitor species"

  • These expression differences likely influence downstream processes that may interact with or be affected by ndhH function

• Novel gene expression:

  • "Both S. officinarum and the hybrid had an abundance of novel genes like sugar phosphate translocator, while S. spontaneum had just one"

  • This indicates that hybridization can lead to the enhanced expression of certain genes that may not be highly expressed in one or both parents

These findings highlight how interspecific hybridization has shaped gene expression patterns in Saccharum hybrids, including genes related to chloroplast function like ndhH, potentially contributing to the superior agronomic traits of these hybrids.

How does the sequence and function of ndhH vary across different Saccharum species and what are the implications for hybrids?

The sequence and function of ndhH show interesting variations across different Saccharum species, with important implications for hybrids derived from these species:

The variations in ndhH across Saccharum species represent both evolutionary history and functional adaptation, providing valuable genetic resources for the improvement of sugarcane hybrids through traditional breeding or genetic engineering approaches.

How can antibodies against ndhH be used for research on Saccharum hybrids?

Antibodies against ndhH provide powerful tools for studying this protein in Saccharum hybrids. Here are specific methodological approaches for their application in research:

• Western blot analysis:

  • Anti-ndhH antibodies can detect the protein in plant extracts with high specificity

  • Recommended protocol: Use a dilution of 1:5000 for optimal results with purified antibodies

  • The expected molecular weight of ndhH is approximately 49 kDa, though apparent molecular weight on SDS-PAGE may be around 45-49 kDa

  • Sample preparation: Total proteins from Saccharum leaves can be precipitated with 10% TCA, washed with acetone, and solubilized in a buffer containing 8M urea, 100 mM Tris-HCL pH 7.5, 1 mM EDTA, and 2% SDS

• Immunolocalization studies:

  • Antibodies can determine the subcellular localization of ndhH within plant tissues

  • For chloroplast proteins, techniques like immunogold electron microscopy provide high-resolution localization

  • This can reveal whether ndhH distribution differs between Saccharum hybrids and their progenitor species

• Protein quantification:

  • Compare ndhH levels between different Saccharum cultivars or under different environmental conditions

  • Normalize using appropriate housekeeping proteins as loading controls

  • This approach can identify cultivars with enhanced expression, potentially correlating with improved photosynthetic efficiency

• Protein-protein interaction studies:

  • Use anti-ndhH antibodies for co-immunoprecipitation experiments

  • Identify proteins that interact with ndhH, potentially revealing novel components of the NAD(P)H-quinone oxidoreductase complex

  • Compare interaction partners between different Saccharum hybrids and progenitor species

• Validating gene editing results:

  • Confirm the effects of CRISPR/Cas9 or other gene editing approaches on ndhH protein levels

  • Essential for connecting genetic modifications to phenotypic changes

When using antibodies, validation is critical as reactivity can vary between species. While some anti-ndhH antibodies have confirmed reactivity with Arabidopsis thaliana, Setaria viridis, and Zea mays , their specificity for Saccharum hybrids should be verified before extensive use. Cross-reactivity testing with proteins from different Saccharum species can provide insights into structural conservation of the ndhH protein.

What methodological approaches can be used to study the involvement of ndhH in stress responses in Saccharum hybrids?

Investigating the role of ndhH in stress responses in Saccharum hybrids requires a multi-faceted methodological approach:

• Experimental design for stress treatments:

  • Implement randomized block designs to account for variation between Saccharum cultivars

  • Include appropriate controls and multiple biological replicates

  • Apply graduated stress levels (e.g., control, mild stress, severe stress) as used in previous studies with Saccharum hybrids

  • Monitor physiological parameters to quantify stress severity

• Transcriptional analysis:

  • Use RNA-seq to quantify ndhH expression under different stress conditions

  • Long read sequencing technology is particularly valuable for the complex Saccharum genome to capture full-length transcripts without assembly artifacts

  • Compare expression patterns across different tissues (e.g., leaf and root) during stress exposure

  • Analyze multiple time points to capture the dynamics of the stress response

• Protein-level analysis:

  • Western blotting with anti-ndhH antibodies to quantify protein levels under stress

  • Investigate post-translational modifications that might regulate ndhH function during stress

  • Examine the assembly and stability of the NAD(P)H-quinone oxidoreductase complex

• Photosynthetic performance assessment:

  • Measure chlorophyll fluorescence parameters to assess photosystem II efficiency

  • Analyze electron transport rates and cyclic electron flow, which involve the NAD(P)H-quinone oxidoreductase complex

  • Compare gas exchange parameters under stress conditions

• Comparative analysis across genotypes:

  • Study ndhH responses in different Saccharum hybrids with varying stress tolerance

  • Include progenitor species (S. officinarum and S. spontaneum) in the analysis

  • Given that "stress and senescence related transcripts were from S. spontaneum in the hybrid" , examine whether ndhH responses reflect this pattern

• Integration with systems biology approaches:

  • Correlate ndhH expression/activity with global transcriptome, proteome, and metabolome changes

  • Identify regulatory networks involving ndhH under stress conditions

  • Use pathway analysis to determine how ndhH function connects to broader stress response mechanisms

By combining these methodological approaches, researchers can develop a comprehensive understanding of how ndhH contributes to stress responses in Saccharum hybrids, potentially identifying targets for improving stress tolerance in these economically important crops.

How can gene editing techniques be applied to study the function of ndhH in Saccharum hybrids?

Gene editing technologies offer powerful approaches to study ndhH function in Saccharum hybrids, though they present unique challenges given the complex polyploid genome of these plants. Here's a methodological framework:

• Target site selection and gRNA design:

  • Analyze the ndhH sequence across all homeologous copies in the Saccharum hybrid genome

  • Design guide RNAs that target conserved regions across homeologous copies

  • Use computational tools to minimize off-target effects

  • Consider targeting different functional domains to create a range of mutations with varying phenotypic effects

• Transformation and delivery strategies:

  • Develop efficient protocols for Saccharum transformation, which can be challenging due to its recalcitrant nature

  • Options include Agrobacterium-mediated transformation, biolistic methods, or protoplast transformation

  • For chloroplast genes like ndhH, consider plastid transformation methods that directly target the chloroplast genome

  • Use tissue-specific or inducible promoters to control Cas9 expression

• Mutation verification methods:

  • Screen putative mutants using PCR amplification and sequencing

  • Verify changes at the protein level using Western blotting with anti-ndhH antibodies

  • For chloroplast genes, determine the homoplasmicity (complete replacement of all wild-type copies) versus heteroplasmicity (mixture of wild-type and mutant copies)

  • Whole genome or plastome sequencing to confirm target mutations and check for off-target effects

• Functional characterization approaches:

  • Analyze photosynthetic parameters using techniques like chlorophyll fluorescence and gas exchange measurements

  • Compare growth and development under different environmental conditions, particularly under stress

  • Examine the assembly and activity of the NAD(P)H-quinone oxidoreductase complex

  • Study responses to environmental stresses like drought, high light, or temperature extremes

• Advanced phenotyping:

  • Use high-throughput phenotyping platforms to capture subtle phenotypic differences

  • Examine field performance of mutants compared to wild-type plants

  • Monitor sugar content and biomass accumulation, as these are key traits in Saccharum hybrids

Gene editing approaches have already shown success in sugarcane: "There have been successful experiments in gene editing in sugarcane, leading to modified/altered sugar and biomass compositions" . These techniques could be adapted to study ndhH function, potentially leading to insights that could improve photosynthetic efficiency and stress tolerance in these important crops.

What does chloroplast genome analysis reveal about the evolutionary history of ndhH in Saccharum hybrids?

Chloroplast genome analysis provides crucial insights into the evolutionary history of ndhH and its significance in Saccharum hybrids:

• Maternal inheritance patterns:

  • The chloroplast genome in Saccharum hybrids is maternally inherited, typically from S. officinarum in commercial hybrids

  • This inheritance pattern explains why "the hybrid shared a larger number of transcripts with S. officinarum than with S. spontaneum" for chloroplast genes

  • Understanding this pattern is essential for interpreting ndhH function in the hybrid genetic background

• Structural genome differences:

  • Comparative analysis revealed "a notable difference in the LSC region of wild and cultivated sugarcanes"

  • "Chloroplasts of sugarcane cultivars showed a loss of a duplicated fragment with 1,031 bp in the beginning of the LSC region, which decreased the chloroplast gene content in hybrids"

  • While this specific difference doesn't directly affect ndhH, it demonstrates how hybridization can lead to structural changes in the chloroplast genome

• Conservation of boundary regions:

  • The location of ndhH at the junction of SSC and IR regions is conserved across Saccharum species

  • "Both the ndhH and ndhF genes located on the JSA (SSC/IRa) and JSB (SSC/IRb)"

  • This conservation suggests functional importance of maintaining these genomic arrangements

• Phylogenetic relationships:

  • Analysis of chloroplast genes including ndhH helps establish evolutionary relationships among Saccharum species

  • Comparison of complete chloroplast genomes has facilitated the development of a robust phylogeny of the Saccharum complex

  • These analyses confirm that modern sugarcane hybrids are more closely related to S. officinarum than to S. spontaneum at the chloroplast level

• Implications for hybrid breeding:

  • The close relationship between hybrid chloroplast genomes and S. officinarum suggests that introducing chloroplast diversity from other sources might benefit breeding programs

  • This has led to efforts to develop "Saccharum hybrids with the cytoplasm of S. spontaneum for breeding purpose"

  • Such cytoplasmic diversity could introduce novel ndhH variants with potential adaptive advantages

The comparative analysis of chloroplast genomes, including ndhH, serves as "a very important tool for deciphering and understanding hybrid Saccharum lineages" . This knowledge not only illuminates evolutionary history but also guides breeding strategies aimed at diversifying the genetic base of commercial sugarcane.