Recombinant Arabidopsis thaliana NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3-B (At1g14450)

What is At1g14450 and what is its basic function?

At1g14450 encodes a NADH dehydrogenase (ubiquinone) protein that functions as part of the electron transport machinery in Arabidopsis thaliana. According to the Araport11 annotation, it's specifically classified as a NADH dehydrogenase (ubiquinone) component . This protein likely contributes to oxidative phosphorylation processes by facilitating electron transfer from NADH to ubiquinone. The gene is located on chromosome 1 of the Arabidopsis genome and produces a protein product that integrates into multi-subunit complexes involved in cellular energy production. Understanding this basic function provides the foundation for more detailed investigations into its specific roles in plant metabolism and stress responses, particularly in relation to photosynthetic and respiratory electron transport chains.

What genetic resources are available for studying At1g14450?

Several genetic resources are available for studying At1g14450, with T-DNA insertion lines being particularly valuable. The SAIL_904_H11 line (stock number CS840866) represents a well-characterized T-DNA insertion mutant available from the Arabidopsis Biological Resource Center (ABRC) . This line was generated by vacuum infiltration of Columbia (Col) ecotype plants with Agrobacterium tumefaciens vector pDAP101, and carries BASTA resistance as a selectable marker . Researchers should note that this line may contain additional insertions potentially segregating in the population, and phenotypes unlinked to the At1g14450 disruption may be present . Quality control data from the J. Ecker lab at SALK Institute has identified additional T-DNA insertions in this stock through next-generation sequencing (TDNA-Seq) . When designing experiments with these resources, researchers should include appropriate genotyping to confirm insertion presence and zygosity, and consider complementation studies to verify that observed phenotypes are specifically linked to At1g14450 disruption.

How does At1g14450 relate to NDH complex function in chloroplasts?

While specific information about At1g14450's direct role in NDH complexes isn't detailed in the provided sources, we can draw meaningful parallels based on our understanding of NDH complex components. The chloroplast NADH dehydrogenase-like (NDH) complex plays critical roles in cyclic electron transport around photosystem I (PSI) and chlororespiration . Like other NDH subunits such as NdhV discussed in research literature, At1g14450 likely contributes to the stability and function of this complex. The fragile nature of NDH complexes, which readily disassociate in the presence of detergents, salt, and alkaline solutions, suggests that peripheral subunits like At1g14450 may play important roles in maintaining structural integrity . The study of At1g14450 would benefit from approaches similar to those used for other NDH subunits, including analysis of mutant phenotypes under varying light conditions, examination of complex assembly via blue native PAGE, and investigation of electron transport rates through spectroscopic methods.

What experimental approaches are suitable for basic characterization of At1g14450?

For basic characterization of At1g14450, researchers should employ a multi-faceted approach combining molecular, biochemical, and physiological techniques. Initial characterization should include gene expression analysis using RT-qPCR to determine tissue-specific expression patterns and responses to environmental stimuli. Protein localization studies using GFP fusion constructs or immunolocalization with specific antibodies can establish the subcellular distribution of the At1g14450 gene product. Phenotypic analysis of T-DNA insertion mutants like SAIL_904_H11 (CS840866) under various growth conditions will reveal functional impacts of gene disruption. Basic biochemical characterization should include blue-native PAGE combined with immunodetection using specific antibodies, similar to approaches used for other NDH complex components . For physiological characterization, measurements of cyclic electron flow around PSI using chlorophyll fluorescence and P700 absorbance changes will help establish the protein's role in photosynthetic electron transport. This systematic characterization will provide a solid foundation for more advanced functional studies.

How can I express and purify recombinant At1g14450 protein for functional studies?

Expression and purification of recombinant At1g14450 requires careful optimization due to the often challenging nature of membrane-associated proteins. Based on established protocols for similar proteins, I recommend a Gateway cloning approach similar to that used for other NDH subunits . First, amplify the full-length At1g14450 coding sequence using high-fidelity polymerase and clone it into pDONR207 entry vector. Transfer the sequence to an appropriate destination vector like pDEST15 for N-terminal GST fusion, which facilitates purification while potentially enhancing solubility . For expression, use E. coli BL21 pLysS cells grown at lower temperatures (16-18°C) after IPTG induction to reduce inclusion body formation. Following cell lysis, purify the protein using GST-Sepharose affinity chromatography with careful optimization of buffer conditions to maintain protein stability . If the full-length protein proves difficult to express, consider designing constructs expressing hydrophilic domains while excluding transmembrane regions, as was successful for other NDH subunits (e.g., 18-kDa subunit Glu37–Asn154, MWFE Met22–Ser65) . Verify purified protein identity using mass spectrometry and assess functionality through in vitro enzyme activity assays appropriate for NADH dehydrogenase components.

What approaches are effective for studying At1g14450 protein interactions within the NDH complex?

Studying At1g14450 protein interactions within the NDH complex requires methods that preserve the integrity of these fragile multi-protein assemblies. Blue-Native PAGE (BN-PAGE) provides an excellent starting point, allowing separation of intact protein complexes combined with immunodetection using antibodies against At1g14450 and known NDH subunits . For more comprehensive interaction studies, chemical crosslinking followed by co-immunopurification offers powerful insights, as demonstrated with other NDH subunits . This approach involves treating isolated chloroplasts or thylakoids with membrane-permeable crosslinkers like DSP (dithiobis[succinimidylpropionate]), followed by solubilization and immunoprecipitation using anti-At1g14450 antibodies. The crosslinked proteins can then be identified through mass spectrometry analysis . For quantitative assessment of protein stoichiometry within the complex, combine immunoblotting of purified complexes with known protein standards, similar to the approach that revealed equimolar concentrations of NdhV and NdhN . For dynamic interaction studies, consider split-fluorescent protein assays in plant protoplasts, allowing visualization of interactions in near-native conditions. These complementary approaches will provide a comprehensive map of At1g14450's position and role within the NDH complex architecture.

How does light intensity affect At1g14450 expression and NDH complex stability?

Light intensity significantly influences NDH complex stability and potentially At1g14450 expression, based on studies of similar components. To investigate this relationship, design experiments that expose wild-type and At1g14450 mutant plants (such as SAIL_904_H11/CS840866) to varying light intensities ranging from low light (50-100 μmol photons m⁻² s⁻¹) to high light (800-1000 μmol photons m⁻² s⁻¹) conditions. Based on findings with other NDH subunits like NdhV, NDH subcomplexes are more rapidly degraded under high-light treatment in subunit-deficient mutants compared to wild type . For expression analysis, collect leaf tissue at regular intervals (0, 2, 6, 12, 24 hours) after light treatment and perform RT-qPCR to quantify At1g14450 transcript levels. In parallel, isolate thylakoid membranes for protein analysis using both denaturing SDS-PAGE and blue-native PAGE combined with immunodetection to assess both protein abundance and complex integrity . For functional assessment, measure NDH activity using in-gel nitro blue tetrazolium (NBT) reduction assays and chlorophyll fluorescence analysis to detect post-illumination increases in fluorescence that indicate NDH-dependent cyclic electron flow . Compare degradation rates of NDH subcomplex A (SubA) and subcomplex E (SubE) between wild-type and At1g14450 mutant plants to determine if At1g14450, like NdhV, plays a role in stabilizing these subcomplexes under high-light stress .

What methods are optimal for analyzing At1g14450 post-translational modifications?

Analysis of post-translational modifications (PTMs) in At1g14450 requires a systematic approach combining enrichment strategies with high-resolution mass spectrometry. Begin with protein extraction from Arabidopsis thylakoid membranes using detergent solubilization with dodecylmaltoside (1% w/v) in aminocaproic acid buffer . After separation of membrane and soluble fractions by centrifugation, perform acetone precipitation followed by tryptic digestion in 50 mM NH₄HCO₃ with DTT reduction and iodoacetamide alkylation . For phosphorylation analysis, enrich phosphopeptides using titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC). For redox modifications, consider differential alkylation approaches to capture reversible cysteine oxidation. Analyze the enriched peptides using LC-MS/MS on a high-resolution instrument such as a Q-TOF mass spectrometer with an HPLC Chip Cube source, similar to the system described for other NDH subunits . Run samples through a 40-nl enrichment column and a 150-mm separation column with a 1-hour gradient of 5-60% acetonitrile containing 0.1% formic acid . Analyze the resulting spectra using database search algorithms that account for variable modifications of interest. Compare PTM profiles between different light conditions, developmental stages, or stress treatments to identify regulatory modifications that might influence At1g14450 function within the NDH complex.

What experimental design is appropriate for analyzing At1g14450 function in developmental contexts?

To analyze At1g14450 function across developmental contexts, implement a multifaceted experimental design that captures spatial, temporal, and condition-dependent aspects of gene function. Begin with a developmental series experiment examining At1g14450 expression from seed germination through flowering using both transcript analysis (RT-qPCR) and protein detection (western blotting with specific antibodies). Compare wild-type plants with homozygous T-DNA insertion mutants (SAIL_904_H11/CS840866) across this developmental series, documenting phenotypic differences in growth rates, photosynthetic parameters, and stress responses. For spatial analysis, employ promoter:GUS fusion constructs to visualize tissue-specific expression patterns, complemented by cell-type specific transcriptomics using fluorescence-activated cell sorting (FACS) or laser-capture microdissection. For functional complementation, create transgenic lines expressing At1g14450 under both native and constitutive promoters in the T-DNA mutant background. Include developmental stage-specific inducible expression systems (such as estradiol-inducible promoters) to determine critical developmental windows for At1g14450 function. Assess NDH complex integrity at each developmental stage using blue-native PAGE combined with immunodetection and measure NDH activity through chlorophyll fluorescence analysis. This comprehensive approach will reveal when and where At1g14450 functions during plant development and how its absence affects growth and photosynthetic efficiency across the plant life cycle.

How should I design experiments to distinguish At1g14450's role in different cellular compartments?

Distinguishing At1g14450's role in different cellular compartments requires a carefully designed experimental approach combining subcellular fractionation, localization studies, and compartment-specific functional assays. First, perform subcellular fractionation to separate chloroplasts, mitochondria, and other cellular components through differential centrifugation. Analyze each fraction by immunoblotting using antibodies against At1g14450 alongside marker proteins for each compartment . For higher resolution localization, create fluorescent protein fusions (both N- and C-terminal) with full-length At1g14450 and potential targeting sequences identified through bioinformatic analysis. Express these constructs in Arabidopsis protoplasts and stable transgenic plants for confocal microscopy visualization. To address potential dual targeting, implement a protein complementation approach using split fluorescent proteins targeted to different compartments. For functional analysis in specific compartments, create constructs with compartment-specific targeting sequences fused to At1g14450 coding sequence and express these in the SAIL_904_H11 (CS840866) mutant background . Assess restoration of wild-type phenotypes through growth analysis, electron transport measurements (using compartment-specific substrates and inhibitors), and complex assembly via blue-native PAGE . Include biochemical confirmation through protocol-specific isolation of protein complexes from each compartment followed by mass spectrometry analysis to identify At1g14450 interaction partners . This integrated approach will precisely define the subcellular distribution of At1g14450 and its functional significance in each compartment.

How can I design experiments to assess At1g14450 function under different stress conditions?

Designing experiments to assess At1g14450 function under stress conditions requires a systematic approach comparing wild-type and mutant responses across multiple stress types and intensities. Begin with a factorial experimental design exposing wild-type Columbia plants and At1g14450 T-DNA insertion mutants (SAIL_904_H11/CS840866) to combinations of abiotic stresses including high light (800-1000 μmol photons m⁻² s⁻¹), drought (withholding water until 30% relative soil water content), cold (4°C), heat (38°C), and nutrient limitation. Monitor physiological parameters including photosynthetic efficiency (Fv/Fm, ΦPSII), growth rates, and visible stress symptoms. For molecular analysis, collect tissue samples at multiple time points (0, 3, 6, 12, 24, 48 hours) after stress initiation to capture both early signaling and later acclimation responses. Analyze At1g14450 expression using RT-qPCR and protein levels using immunoblotting with specific antibodies. Assess NDH complex integrity under each stress condition using blue-native PAGE combined with immunodetection of NDH subunits , with particular attention to subcomplexes A and E which may show differential stability in the absence of stabilizing subunits . Measure NDH activity through post-illumination chlorophyll fluorescence rise and in-gel NBT reduction assays . Include complementation lines expressing At1g14450 under both constitutive and stress-inducible promoters to determine if targeted expression can enhance stress tolerance. This comprehensive approach will reveal how At1g14450 contributes to stress responses and whether its function becomes particularly critical under specific stress conditions.

What is the optimal approach for generating and validating antibodies against At1g14450?

Generating and validating antibodies against At1g14450 requires careful antigen design and comprehensive validation. Begin with epitope prediction analysis to identify immunogenic regions that are accessible in the native protein and unique compared to other Arabidopsis proteins. Consider producing antibodies against both the full-length protein and specific peptide sequences. For recombinant protein production, clone the At1g14450 coding sequence (or hydrophilic domains if membrane-spanning regions are present) into an expression vector such as pDEST15 for N-terminal GST fusion . Express the fusion protein in E. coli BL21 pLysS cells and purify using GST-Sepharose affinity chromatography . For immunization, inject purified protein into rabbits following a protocol similar to that used for other NDH subunits (four doses of 250 μg per protein) . Collect serum after the third and fourth injections for validation. Validation must include multiple controls: (1) western blot comparison of wild-type versus At1g14450 knockout mutant (SAIL_904_H11/CS840866) proteins; (2) preimmune serum controls; (3) peptide competition assays; (4) detection of recombinant At1g14450 protein; and (5) immunoprecipitation followed by mass spectrometry to confirm specificity. Optimize antibody working dilutions for different applications (western blotting, immunoprecipitation, immunolocalization) using titration experiments. Consider affinity purification of antibodies against the immunizing antigen to improve specificity. Once validated, these antibodies will be invaluable tools for studying At1g14450 expression, localization, and protein interactions across different experimental conditions.

How should I interpret conflicting phenotypes in different At1g14450 mutant lines?

Interpreting conflicting phenotypes between different At1g14450 mutant lines requires a systematic approach to identify the source of variation. First, thoroughly genotype all mutant lines using PCR to confirm the exact position and nature of each mutation, and verify that no wild-type transcript is produced through RT-PCR. For T-DNA insertion lines like SAIL_904_H11 (CS840866) , confirm insertion homozygosity and determine whether truncated transcripts might be produced. Second, characterize each line's genetic background, recognizing that T-DNA lines may contain additional insertions potentially segregating and causing unlinked phenotypes . The SAIL_904_H11 line specifically has been identified to contain additional T-DNA insertions through next-generation sequencing (TDNA-Seq) . Third, systematically compare growth conditions between experiments, as phenotypes related to NDH function often manifest differently under varying light intensities and other environmental factors . Fourth, perform allelism tests by crossing different mutant lines and analyzing F1 and F2 generations. If the mutations affect the same gene, trans-heterozygotes should display the mutant phenotype. Fifth, conduct complementation studies by transforming each mutant line with the wild-type At1g14450 gene under its native promoter. Resolution of the phenotype confirms that disruption of At1g14450 (rather than background mutations) causes the observed effects. Finally, create a detailed phenotypic profile of each line across multiple parameters including growth metrics, photosynthetic efficiency, stress responses, and biochemical characteristics of the NDH complex to identify subtle differences that might explain the conflicting observations.

How do I integrate proteomics and transcriptomic data to understand At1g14450 function?

Integrating proteomics and transcriptomics data requires a multi-stage analytical pipeline to comprehensively understand At1g14450 function. Begin by designing experiments that collect matched samples for both RNA-seq and proteomics analysis from identical tissues under the same conditions, comparing wild-type and At1g14450 mutant (SAIL_904_H11/CS840866) plants. For proteomics, implement both soluble protein extraction and membrane protein isolation using dodecylmaltoside solubilization , followed by both shotgun proteomics and targeted analysis of protein complexes using blue-native PAGE. Process proteomic samples using tryptic digestion with DTT reduction and iodoacetamide alkylation, followed by LC-MS/MS analysis . For data integration, first normalize and analyze each dataset independently using appropriate statistical methods, identifying differentially expressed genes and proteins. Then implement multi-omics integration using several complementary approaches: (1) direct correlation analysis between transcript and protein levels for individual genes; (2) pathway enrichment analysis using tools like GSEA to identify consistently affected biological processes; (3) protein complex analysis focusing specifically on NDH complex components and their transcriptional and post-transcriptional regulation; (4) regulatory network reconstruction using tools like WGCNA to identify co-regulated modules; and (5) causal network analysis to distinguish primary from secondary effects of At1g14450 disruption. Visualize integrated results using tools like Cytoscape for network representation and heatmaps for expression patterns. This systematic integration will reveal how transcriptional and post-transcriptional mechanisms coordinate At1g14450 function and identify compensatory responses activated in mutant plants.

How can I distinguish between direct and indirect effects of At1g14450 mutation on photosynthetic parameters?

Distinguishing between direct and indirect effects of At1g14450 mutation on photosynthetic parameters requires a multifaceted experimental approach combined with careful data interpretation. First, establish a temporal sequence by monitoring photosynthetic parameters and gene expression changes at multiple time points following controlled induction of At1g14450 disruption using an inducible gene silencing system (such as dexamethasone-inducible RNAi). Early changes (minutes to hours) likely represent direct effects, while later changes (days) may reflect indirect adaptations. Second, create a series of double mutants combining At1g14450 mutation (SAIL_904_H11/CS840866) with mutations in other photosynthetic complexes to identify epistatic relationships. Third, perform detailed biochemical analysis of specific electron transport components using isolated thylakoid membranes with defined electron donors and acceptors to pinpoint exactly which reactions are altered in the mutant. Fourth, use protein-protein interaction studies (co-immunoprecipitation or crosslinking combined with mass spectrometry) to identify direct interaction partners of At1g14450, which would likely be directly affected by its absence. Fifth, implement comparative analysis across different environmental conditions, as direct effects should be consistently observed while indirect compensatory responses may vary. Sixth, perform transcriptome analysis of wild-type and mutant plants to identify primary and secondary response genes using network analysis tools that distinguish direct regulatory targets from downstream effects. Finally, create complementation lines with inducible expression of At1g14450 in the mutant background and monitor the kinetics of phenotype rescue, as direct effects should be rapidly restored. This systematic approach will create a causal model distinguishing primary consequences of At1g14450 absence from secondary adaptive responses.

What bioinformatics tools are most useful for structural prediction and functional annotation of At1g14450?

For comprehensive structural prediction and functional annotation of At1g14450, a multi-layered bioinformatics approach utilizing the latest tools is essential. Begin with primary sequence analysis using InterProScan to identify conserved domains, motifs, and functional sites, complemented by transmembrane topology prediction using TMHMM and signal peptide analysis with SignalP. For evolutionary context, perform comprehensive ortholog identification using OrthoFinder across diverse plant species, followed by multiple sequence alignment with MAFFT and phylogenetic analysis using IQ-TREE to identify conserved regions under selection pressure. For structural prediction, implement AlphaFold2, which has revolutionized protein structure prediction with near-experimental accuracy, especially for soluble domains. For membrane-spanning regions, use specialized tools like AlphaFold-Multimer in combination with molecular dynamics simulations to refine predictions in a membrane environment. To predict protein-protein interactions within the NDH complex, use both computational approaches (STITCH, STRING) and co-evolution analysis (EVcouplings) to identify potential interaction interfaces. For functional annotation, implement Gene Ontology enrichment analysis using tools like PANTHER, complemented by literature-derived function prediction with PubTator. Integrate these predictions with published experimental data on NDH complex assembly and function to develop testable hypotheses about At1g14450's specific role. For visualization and analysis of predicted structures, use PyMOL to map conserved residues, potential post-translational modification sites, and predicted interaction interfaces onto the three-dimensional model. This comprehensive bioinformatics approach will generate structurally informed hypotheses about At1g14450 function that can guide experimental design.

What is the most efficient protocol for genotyping At1g14450 T-DNA insertion mutants?

The most efficient protocol for genotyping At1g14450 T-DNA insertion mutants combines PCR-based screening with quality control measures to ensure accurate genotyping. For the SAIL_904_H11 (CS840866) line , design three PCR primers: two gene-specific primers flanking the insertion site (LP and RP) and one T-DNA border-specific primer (BP). The optimal primer design should position LP and RP approximately 900-1000 bp apart for wild-type amplification, with primers having similar melting temperatures (Tm ≈ 60°C) and GC content between 40-60%. Extract genomic DNA from young leaf tissue using a rapid CTAB or NaOH-based extraction method. Set up two PCR reactions for each plant: LP+RP to amplify the wild-type allele and BP+RP to detect the T-DNA insertion. Optimize PCR conditions using touchdown PCR (starting 5°C above calculated Tm and decreasing by 0.5°C per cycle for 10 cycles) followed by 25 regular cycles, which reduces non-specific amplification. Wild-type plants will show only the LP+RP product, homozygous mutants will show only the BP+RP product, and heterozygotes will show both bands. For quality control, include known wild-type and homozygous mutant samples as controls in each PCR run. For high-throughput screening, implement multiplexed PCR with all three primers in a single reaction with differentially sized products. Verify insertion positions through sequencing of BP+RP products to confirm the exact insertion site, especially important as TDNA-Seq has identified additional T-DNA insertions in this stock . This protocol ensures reliable identification of homozygous mutants for subsequent functional studies.

How should I optimize Blue-Native PAGE to effectively analyze At1g14450-containing complexes?

Optimizing Blue-Native PAGE (BN-PAGE) for At1g14450-containing complexes requires careful attention to sample preparation and electrophoresis conditions to preserve these fragile assemblies. Begin with freshly isolated thylakoid membranes from young Arabidopsis leaves, keeping all steps at 4°C to minimize proteolysis. Solubilize membranes using mild detergents - start with 1% (w/v) dodecylmaltoside in ACA buffer (750 mM aminocaproic acid, 0.5 mM EDTA, 50 mM Tris-HCl, pH 7.0) , but optimize detergent concentrations (0.5-1.5%) based on complex integrity. After solubilization, incubate samples for exactly 20 minutes at 4°C followed by centrifugation at 20,000 × g for 10 minutes . Add Serva Blue G (0.2% v/v final concentration) to the supernatant just before loading . Prepare gradient gels (4.5-16% w/v) with acrylamide:bisacrylamide ratios of 32:1 for optimal resolution of large complexes. Run the first dimension at 4°C using cathode buffer containing Serva Blue G for the first third of the run, then switch to cathode buffer without dye to improve resolution. For highest resolution, limit protein loading to 100 μg per lane and use moderate voltage (100V) throughout the run. For effective visualization of At1g14450-containing complexes, transfer proteins to PVDF membranes and probe with specific antibodies. For activity staining, implement in-gel nitro blue tetrazolium (NBT) reduction assays immediately after the first dimension . For subcomplex analysis, excise lanes from the first dimension, incubate in denaturing buffer, and run a second dimension SDS-PAGE followed by immunoblotting or mass spectrometry. Include molecular weight markers (66–669 kDa) to estimate complex sizes accurately.

What is the optimal protocol for measuring NDH complex activity in At1g14450 mutants?

The optimal protocol for measuring NDH complex activity in At1g14450 mutants combines multiple complementary approaches to provide comprehensive functional assessment. Begin with in vivo measurements using chlorophyll fluorescence analysis on dark-adapted leaves. Measure the post-illumination chlorophyll fluorescence rise, which reflects NDH-mediated cyclic electron flow, by recording fluorescence transients after switching off actinic light. Quantify both the amplitude and kinetics of this transient increase in wild-type and At1g14450 mutant plants (SAIL_904_H11/CS840866) . For biochemical assessment, isolate thylakoid membranes and implement in-gel activity staining following blue-native PAGE separation. Use nitro blue tetrazolium (NBT) reduction assays with NADH as substrate, which forms purple formazan deposits at the position of active NDH complex. Quantify band intensity using densitometry analysis for comparison between wild-type and mutant samples. For higher sensitivity, implement spectrophotometric assays measuring NADH dehydrogenase activity in solubilized thylakoid preparations using artificial electron acceptors like ferricyanide, with activity monitored as decreasing absorbance at 340 nm (NADH oxidation). To assess cyclic electron flow around PSI, measure P700 redox kinetics using dual-wavelength (820/870 nm) spectroscopy, focusing on the re-reduction rate of P700+ after a light-to-dark transition, which reflects cyclic electron flow capacity. Perform all measurements under varying light intensities and following different light treatments, as NDH activity and its mutant phenotypes become more pronounced under specific conditions, particularly high light stress . Include known NDH mutants like ndhv as positive controls, as these show impaired NDH activity and can serve as reference points for phenotype severity .

How can I effectively characterize At1g14450 expression patterns at both transcript and protein levels?

Effectively characterizing At1g14450 expression requires a comprehensive approach integrating transcript and protein analyses across tissues, developmental stages, and environmental conditions. For transcript analysis, implement RT-qPCR using carefully designed primers spanning exon-exon junctions to ensure specific amplification of mature mRNA. Validate primer efficiency (90-110%) and specificity (single melting peak) before experimental use. Use multiple reference genes validated for stability across your experimental conditions for accurate normalization. Complement qPCR with RNA-seq for genome-wide context and detection of alternative splicing variants. For spatial resolution of expression, create promoter:GUS fusion constructs incorporating approximately 2 kb of sequence upstream of the At1g14450 start codon, and analyze GUS activity histochemically across tissues and developmental stages. For protein-level analysis, generate specific antibodies against At1g14450 following protocols similar to those used for other NDH subunits, using purified recombinant protein for immunization . Validate antibody specificity using wild-type versus mutant (SAIL_904_H11/CS840866) comparison. Perform western blot analysis of protein extracts from different tissues, developmental stages, and plants exposed to varying environmental conditions. For subcellular localization, perform immunogold labeling for transmission electron microscopy or create fluorescent protein fusions for confocal microscopy. For turnover and stability assessment, conduct cycloheximide chase experiments combined with western blotting to determine protein half-life. Integrate these approaches to create a comprehensive expression atlas of At1g14450, identifying conditions where its expression is particularly critical and revealing potential regulatory mechanisms controlling its abundance at both transcriptional and post-transcriptional levels.

What approaches can determine whether At1g14450 undergoes post-translational modifications?

Determining whether At1g14450 undergoes post-translational modifications (PTMs) requires a systematic multi-method approach focusing on various modification types. Begin with bioinformatic prediction using tools like NetPhos, UbPred, and SUMOplot to identify potential modification sites based on sequence motifs. For experimental validation, implement a comprehensive mass spectrometry-based approach. First, enrich At1g14450 through immunoprecipitation using specific antibodies or express tagged versions of the protein for affinity purification. Process samples following protocols similar to those used for other NDH subunits: reduce with DTT, alkylate with iodoacetamide, and digest with trypsin for 16 hours at 37°C . For phosphorylation analysis, enrich phosphopeptides using titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC) before LC-MS/MS analysis. Analyze samples on a high-resolution mass spectrometer (e.g., Q-TOF) with an HPLC Chip Cube source , using both data-dependent acquisition for discovery and parallel reaction monitoring for targeted quantification of specific modifications. For redox modifications, implement differential alkylation strategies to distinguish reduced from oxidized cysteines. For glycosylation, use specific glycosidases followed by mobility shift analysis on SDS-PAGE. For ubiquitination and SUMOylation, perform western blotting with modification-specific antibodies or express epitope-tagged modifiers (His-ubiquitin or His-SUMO) for affinity purification of modified proteins. Complement these approaches with site-directed mutagenesis of predicted modification sites followed by functional assays to determine their physiological significance. Compare modification patterns between different light conditions, developmental stages, and stress treatments to identify regulatory PTMs that might influence At1g14450 function within the NDH complex.