NRT3.1 Antibody

What is NRT3.1/NAR2.1 and why are antibodies against it important for plant research?

NRT3.1 (also called NAR2.1) is a partner protein that interacts with NRT2 family transporters, particularly NRT2.1, to form functional high-affinity nitrate transport systems in plants. While NRT3.1/NAR2.1 contains only a single transmembrane domain, it is essential for proper localization and function of NRT2 transporters at the plasma membrane . Antibodies against NRT3.1 are critical research tools because:

• They allow detection of the protein in complex samples via western blotting

• They enable investigation of protein-protein interactions

• They permit monitoring of protein levels under different environmental conditions

• They facilitate subcellular localization studies

In Arabidopsis and other plant systems, NRT3.1/NAR2.1 has been demonstrated to be absolutely required for high-affinity nitrate uptake, with nar2.1 null mutants showing strong deficiency in nitrate HATS (High-Affinity Transport System) activity .

How are NRT3.1/NAR2.1 antibodies typically generated for plant research?

NRT3.1/NAR2.1 antibodies are typically generated using the following approaches:

• Peptide-based antibody production: Synthetic peptides corresponding to specific regions of NRT3.1/NAR2.1 are used as antigens. For example:

  • In maize research, anti-NRT3.1A antibody was raised in rabbit against the synthetic peptide (C)LDVTTSAKPGQ

  • For Arabidopsis studies, antibodies have been raised against peptidic sequences within the N-terminus of the protein

• Recombinant protein approach: The full or partial protein is expressed in a bacterial system, purified, and used as an antigen. This approach is often used when larger portions of the protein are needed for antibody generation.

• Purification and validation: After antibody generation, affinity purification is typically performed to enhance specificity, followed by validation in wild-type plants and appropriate mutants (e.g., nar2.1 mutants) to confirm antibody specificity .

The most successful antibodies target unique, accessible epitopes that aren't conserved in related proteins, ensuring specific detection of NRT3.1/NAR2.1.

What are the recommended protein extraction methods for optimal NRT3.1/NAR2.1 detection?

For optimal detection of NRT3.1/NAR2.1 in plant tissues, the following extraction methods are recommended:

Microsomal/Plasma Membrane Preparation:

• Homogenize fresh root tissue (typically 0.5-1g) in extraction buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 250 mM sucrose

  • 10 mM EDTA

  • 10% glycerol

  • Protease inhibitor cocktail

• Centrifuge homogenate at 10,000g to remove cell debris

• Ultracentrifuge supernatant at 100,000g to collect microsomal or plasma membrane fractions

• Resuspend pellet in appropriate buffer containing a mild detergent like 1.5% dodecyl-β-maltoside for blue native gel electrophoresis , or a standard SDS sample buffer for regular SDS-PAGE

Total Protein Extraction:
For detection of total NRT3.1/NAR2.1:

• Homogenize tissue in buffer containing:

  • 1% Nonidet P-40

  • Protease inhibitors

• Clear lysates by centrifugation

• Measure protein concentration

• Analyze samples on 10% SDS-PAGE gels

For detection of native complexes between NRT2.1 and NRT3.1/NAR2.1, membrane solubilization with mild detergents like dodecyl-β-maltoside (1.5%) followed by blue native gel electrophoresis has proven effective .

How can I verify the specificity of my NRT3.1/NAR2.1 antibody?

To verify antibody specificity, implement the following validation steps:

• Genetic controls:

  • Compare western blot signals between wild-type plants and nar2.1 knockout mutants

  • The specific band (approximately 25 kDa for NRT3.1/NAR2.1) should be absent in the mutant

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Apply to duplicate western blots

  • Specific bands should disappear in the peptide-blocked sample

• Mass spectrometry confirmation:

  • Excise the band detected by the antibody

  • Identify the protein by mass spectrometry

  • Confirm it matches NRT3.1/NAR2.1 sequence

  • This approach was used successfully in maize studies, confirming ZmNRT3.1A (ZmNAR2.1) at approximately 21 kDa

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of NRT3.1/NAR2.1

  • Consistent detection patterns increase confidence in specificity

Remember that NRT3.1/NAR2.1 typically appears at approximately 25 kDa in Arabidopsis and about 21 kDa in maize on SDS-PAGE gels.

How can I investigate the NRT2.1-NRT3.1 protein complex using antibody-based approaches?

The NRT2.1-NRT3.1/NAR2.1 complex can be studied using several antibody-based approaches:

• Blue Native PAGE followed by western blotting:

  • Solubilize membranes in 1.5% dodecyl-β-maltoside

  • Separate native complexes by blue native PAGE

  • Perform western blotting with anti-NRT2.1 antibodies

  • An oligomeric complex of approximately 150 kDa has been identified in Arabidopsis

  • For second dimension analysis, excise the lane and perform SDS-PAGE to resolve individual components

• Co-immunoprecipitation (Co-IP):

  • Use either anti-NRT2.1 or anti-NRT3.1 antibodies coupled to a matrix

  • Precipitate the complex from solubilized membranes

  • Analyze precipitated proteins by western blotting with antibodies against both partners

• Proximity-dependent labeling combined with immunoprecipitation:

  • Generate transgenic lines expressing NRT3.1 fused to a biotin ligase

  • Perform proximity labeling followed by streptavidin pulldown

  • Confirm NRT2.1 presence using specific antibodies

Research has shown that the functional complex likely consists of a tetramer containing two subunits each of NRT2.1 and NRT3.1/NAR2.1, with a total molecular mass of approximately 150 kDa . Importantly, monomeric NRT2.1 was absent in functional plants, suggesting the complex rather than the monomer is the active transport unit .

How do NRT3.1/NAR2.1 protein levels correlate with NRT2.1 abundance and high-affinity nitrate uptake?

The relationship between NRT3.1/NAR2.1 protein levels, NRT2.1 abundance, and nitrate uptake activity is complex:

These findings suggest multiple layers of regulation: (1) transcriptional control, (2) protein stability and complex formation, and (3) post-translational regulation of the complex activity, potentially through phosphorylation .

What role does phosphorylation play in regulating the NRT2.1-NRT3.1 complex, and how can it be studied using antibodies?

Phosphorylation is a critical regulatory mechanism for the NRT2.1-NRT3.1 transport system:

• NRT2.1 phosphorylation sites:

  • Phosphoproteomic studies have identified multiple phosphorylation sites in NRT2.1, including S11, S28, S501, and T521

  • The S501 site is particularly important, as mimicking constitutive phosphorylation at this site by substituting with aspartate (S501D) drastically reduces nitrate transport activity

• Methodologies for studying phosphorylation:

  • Phospho-specific antibodies: Antibodies specific to phosphorylated S501 (anti-S501P) have been successfully used to monitor NRT2.1 phosphorylation status

  • ELISA-based quantification: Using both phospho-specific and total protein antibodies enables quantitative assessment of the phosphorylation ratio

  • Western blotting: When using microsomes, NRT2.1 appears in multiple forms (45 kDa monomer, and higher molecular weight forms at ~100 kDa and ~130 kDa), with differential phosphorylation patterns

• Regulation by nitrate:

  • Nitrate re-supply after starvation increases total NRT2.1 protein but decreases the proportion of S501-phosphorylated NRT2.1, particularly in high molecular weight complexes

  • This suggests dephosphorylation of S501 may be required for activation of the transport complex

For studying these phenomena, a combination of phospho-specific antibodies, total protein antibodies, and native protein complex analysis is recommended. Researchers should design experiments that track both total protein levels and phosphorylation status under varying nitrate conditions.

How can I resolve contradictory data between NRT3.1 protein levels and nitrate uptake activity in my research?

When facing discrepancies between NRT3.1/NAR2.1 protein levels and nitrate uptake activity, consider these methodological approaches:

• Examine post-translational modifications:

  • As demonstrated for NRT2.1, phosphorylation can inactivate the transporter without affecting protein levels

  • Investigate NRT3.1/NAR2.1 phosphorylation or other modifications using mass spectrometry or phospho-specific antibodies if available

• Assess complex formation rather than individual proteins:

  • The functional unit appears to be the NRT2.1-NRT3.1 complex rather than individual proteins

  • Use blue native PAGE to determine if complex formation is affected even when individual protein levels remain constant

• Consider compartmentalization:

  • Proteins may be present but not properly localized to the plasma membrane

  • Perform subcellular fractionation to verify proper localization

  • Consider immunolocalization studies to confirm membrane targeting

• Investigate partner protein effects:

  • In studies with transgenic 35S::NRT2.1 plants, despite constitutive NRT2.1 expression, uptake activity was still regulated, suggesting regulation at the NAR2.1 level or through post-translational modifications

  • Examine levels of all transport system components

• Time-resolved measurements:

  • Short-term responses (4h) can differ from long-term responses (24h)

  • Design experiments with multiple timepoints to capture the dynamics of protein levels versus activity

One useful experimental design would be to simultaneously measure: (1) transport activity using 15N-nitrate uptake assays, (2) protein levels by western blotting, (3) complex formation using native PAGE, and (4) post-translational modifications through immunoprecipitation followed by mass spectrometry.

What methodological considerations are important when using anti-NRT3.1 antibodies in heterologous expression systems?

When using anti-NRT3.1 antibodies in heterologous expression systems, consider these critical factors:

• Epitope conservation and antibody specificity:

  • Verify if the epitope recognized by your anti-NRT3.1 antibody is conserved in the heterologous system

  • Perform sequence alignment between your species of interest and the species for which the antibody was developed

  • Consider generating new antibodies if epitope conservation is low

• Positive and negative controls:

  • Include samples from the original species (positive control)

  • Use knockout/mutant samples as negative controls

  • Consider testing the antibody on purified recombinant protein of the heterologous species

• Expression system considerations:

  • Studies have shown that heterologous expression of NRT3.1 from different species (e.g., DsNRT3.1 in Arabidopsis) can affect endogenous nitrate transport systems

  • Monitor potential changes in expression of endogenous NRT transporters (e.g., AtNRT2.1-2.6) when expressing heterologous NRT3.1

  • Verify proper membrane localization using GFP-fusion proteins and confocal microscopy before antibody-based experiments

• Interaction verification:

  • Determine if the heterologous NRT3.1 interacts with endogenous NRT2 family members

  • Use co-immunoprecipitation with antibodies against both the heterologous NRT3.1 and the endogenous NRT2 proteins

  • Consider split-GFP or FRET approaches to verify interactions in vivo

A study demonstrating the importance of these considerations showed that DsNRT3.1 expressed in Arabidopsis affected seedling growth by enhancing nitrate uptake, likely through interaction with endogenous AtNRT2 members, particularly AtNRT2.5 .

How can I optimize western blot conditions for clear detection of NRT3.1/NAR2.1?

Optimizing western blot conditions for NRT3.1/NAR2.1 detection requires attention to several technical details:

• Sample preparation optimizations:

  • Always include protease inhibitors in extraction buffers to prevent degradation

  • For membrane proteins like NRT3.1/NAR2.1, avoid boiling samples if possible; incubation at 37°C for 30 minutes is often sufficient and reduces aggregation

  • Load adequate protein amounts (50-100 μg of total protein or 10-20 μg of membrane protein)

• Gel electrophoresis parameters:

  • Use 10-12% polyacrylamide gels for optimal resolution of NAR2.1/NRT3.1 (~21-25 kDa)

  • Consider gradient gels (4-15%) when analyzing both NRT2.1 (~45 kDa) and NAR2.1 (~25 kDa) in the same sample

  • For native complex analysis, 4-16% gradient blue native gels provide good separation

• Transfer and blocking conditions:

  • Use PVDF membranes for better protein retention and signal-to-noise ratio

  • Optimize transfer conditions: 100V for 1 hour for standard SDS-PAGE or overnight low-voltage transfer for blue native gels

  • Block with 5% non-fat dry milk or 3% BSA in TBS-T

• Antibody dilutions and incubation:

  • Primary antibody dilutions reported in literature:

    • Anti-NRT2.1: 1:2,000

    • Anti-NRT3.1/NAR2.1: 1:2,000

    • Anti-phospho-specific antibodies: 1:1,000

  • Incubate with primary antibody overnight at 4°C for optimal binding

  • Use secondary antibody at 1:10,000 to 1:20,000 dilution

• Detection systems:

  • Enhanced chemiluminescence (ECL) systems offer good sensitivity

  • For quantitative analysis, consider fluorescent secondary antibodies and imaging systems

  • For low abundance detection, use high-sensitivity ECL substrates

If non-specific bands appear, optimize blocking conditions and consider pre-adsorbing the antibody with total protein extract from knockout plants.

What approaches can help identify protein-protein interactions between NRT2 and NRT3.1 proteins using antibodies?

To identify and characterize protein-protein interactions between NRT2 and NRT3.1 proteins:

• Co-immunoprecipitation (Co-IP):

  • Solubilize membranes using mild detergents (1.5% dodecyl-β-maltoside works well)

  • Immunoprecipitate with anti-NRT2.1 or anti-NRT3.1 antibodies

  • Analyze precipitates by western blotting with antibodies against the potential partner proteins

  • Controls should include non-specific IgG and samples from relevant knockout mutants

• Blue native PAGE followed by second-dimension SDS-PAGE:

  • Separate native complexes in the first dimension

  • Cut out the lane and separate component proteins by SDS-PAGE in the second dimension

  • Perform western blotting to identify proteins in the complex

  • This approach revealed an oligomeric complex (~150 kDa) containing both NRT2.1 and NAR2.1 proteins in Arabidopsis

• Crosslinking approaches:

  • Treat intact plant tissues or isolated membranes with membrane-permeable crosslinkers

  • Immunoprecipitate with antibodies against either NRT2.1 or NRT3.1

  • Analyze by mass spectrometry to identify interaction partners

• Proximity-dependent labeling:

  • Express fusion proteins (BioID or TurboID fused to NRT3.1) in plants

  • After biotin treatment, perform streptavidin pulldown

  • Identify proximal proteins using mass spectrometry or western blotting

• FRET/FLIM analysis with antibodies:

  • Use fluorophore-conjugated primary or secondary antibodies against NRT2.1 and NRT3.1

  • Perform FRET analysis on fixed tissue sections

  • This approach requires careful controls and optimization

Research has suggested that the functional unit for high-affinity nitrate influx may be a tetramer consisting of two subunits each of NRT2.1 and NRT3.1/NAR2.1 , highlighting the importance of studying these interactions.

How can I use NRT3.1 antibodies to investigate changes in protein levels in response to environmental stimuli?

To investigate NRT3.1/NAR2.1 protein level changes in response to environmental stimuli:

• Experimental design considerations:

  • Include multiple timepoints to capture rapid (4h) and prolonged (24h) responses

  • Compare protein levels with mRNA expression and transport activity measurements

  • Use consistent growth conditions and treatments across experiments

  • Consider both microsomal fractions and plasma membrane-enriched fractions

• Quantitative western blotting:

  • Include loading controls (H+-ATPase for membrane fractions, actin for total protein)

  • Use standardized amounts of protein (50 μg has been reported as effective)

  • Employ digital imaging systems with linear detection range

  • Analyze band intensity using appropriate software (ImageJ, etc.)

• ELISA-based quantification:

  • Develop sandwich ELISA using anti-NRT3.1 antibodies

  • This approach has been successfully used for quantifying phosphorylated versus total NRT2.1

  • Allows more precise quantification than western blotting

• Experimental treatments to consider:

  • Nitrogen starvation followed by nitrate resupply (1 mM NO3-)

  • High nitrogen (10 mM NH4NO3) exposure

  • Dark/light transitions

  • Treatment with nitrogen metabolites

• Data analysis approaches:

  • Normalize protein levels to appropriate controls

  • Correlate with physiological parameters (root 15NO3- influx)

  • Compare with transcriptional responses

Studies have shown that short-term responses (4h) to high nitrogen or darkness do not significantly affect NRT2.1 and NAR2.1 protein levels despite decreased transport activity, while longer exposures (24h) result in protein level reductions . This highlights the importance of temporal analysis when studying environmental responses.

How can mass spectrometry complement antibody-based approaches for studying NRT3.1 and its interactions?

Mass spectrometry offers powerful complementary approaches to antibody-based methods:

• Identification and validation of antibody targets:

  • Confirm the identity of bands detected by anti-NRT3.1 antibodies

  • This approach has been used to verify the specificity of antibodies against ZmNRT3.1A in maize

  • Provides unambiguous confirmation of antibody specificity

• Post-translational modification mapping:

  • Identify phosphorylation sites on NRT2.1 and potentially on NRT3.1

  • Phosphoproteomic approaches identified S11, S28, S501, and T521 phosphorylation sites on NRT2.1

  • Quantify changes in phosphorylation states under different conditions

• Interactome analysis:

  • Immunoprecipitate NRT3.1 and identify all associated proteins

  • Compare interactomes under different nitrogen conditions

  • Discover novel components of nitrate transport complexes

• Complex stoichiometry determination:

  • Use quantitative proteomics to determine the exact stoichiometry of NRT2.1:NRT3.1 in the transport complex

  • Current evidence suggests a 2:2 ratio in a tetrameric complex , but exact stoichiometry remains to be confirmed

• Selected/Multiple Reaction Monitoring (SRM/MRM):

  • Develop targeted mass spectrometry assays for absolute quantification of NRT3.1

  • This approach can be more precise than antibody-based quantification and doesn't require antibodies

  • Particularly useful for comparing protein levels across different genetic backgrounds or conditions

These mass spectrometry approaches can provide molecular insights that are difficult to obtain through antibody-based methods alone, particularly for understanding the dynamics of complex formation and post-translational modifications.

What are the limitations of current anti-NRT3.1 antibodies and how might they be addressed in future research?

Current anti-NRT3.1 antibodies face several limitations that future research could address:

• Cross-species reactivity limitations:

  • Most antibodies are raised against species-specific epitopes

  • Future development of antibodies targeting highly conserved regions could enable cross-species studies

  • In silico epitope prediction combined with sequence conservation analysis could identify optimal targets

• Inability to distinguish between different complex forms:

  • Current antibodies detect total NRT3.1 but cannot distinguish between monomeric and complexed forms

  • Development of conformation-specific antibodies could enable monitoring of complex assembly/disassembly

• Limited availability of phospho-specific antibodies:

  • While phospho-specific antibodies exist for NRT2.1 (e.g., anti-S501P) , similar tools for NRT3.1 are lacking

  • Phosphoproteomic studies to identify regulatory phosphorylation sites on NRT3.1 followed by phospho-specific antibody development would be valuable

• Technical challenges in membrane protein detection:

  • Membrane proteins like NRT3.1 can be difficult to extract and detect

  • Development of improved extraction protocols specific for NRT3.1 detection could enhance sensitivity

• Future antibody technologies:

  • Single-chain variable fragments (scFvs) or nanobodies against NRT3.1 could provide better access to epitopes in native complexes

  • Bispecific antibodies targeting both NRT2.1 and NRT3.1 might enable specific detection of only the functional complex

  • CRISPR-based epitope tagging of endogenous proteins could overcome antibody specificity issues

• Alternative approaches:

  • Aptamer-based detection methods might offer advantages for conformational studies

  • MS-based absolute quantification could complement or replace antibody-based quantification

  • Fluorescent protein fusions could enable live imaging of complex formation

Addressing these limitations will require interdisciplinary approaches combining structural biology, proteomics, and antibody engineering technologies.

How can NRT3.1 antibodies be applied in conjunction with transcription factor studies to understand nitrogen signaling networks?

Combining NRT3.1 antibody-based approaches with transcription factor studies can provide integrated insights into nitrogen signaling networks:

• Chromatin Immunoprecipitation (ChIP) combined with protein analysis:

  • Use ChIP-qPCR with antibodies against transcription factors like NIGT1 subfamily members to identify direct regulation of NRT3.1 and NRT2 genes

  • Correlate binding events with changes in NRT3.1 protein levels using western blotting

  • This combined approach can link transcriptional regulation to protein expression

• Transcription factor-induced changes in protein complex formation:

  • Analyze how overexpression or knockout of transcription factors affects:

    • NRT2.1 and NRT3.1 protein levels

    • Complex formation between NRT2.1 and NRT3.1

    • Post-translational modifications of these proteins

• Protein-level feedback on transcription:

  • Investigate if changes in NRT3.1/NRT2.1 protein levels or their activity status affect expression of transcription factors

  • Use co-immunoprecipitation to identify potential direct interactions between transporter proteins and signaling components

• Integration with phosphorylation signaling:

  • Monitor how transcription factor manipulation affects phosphorylation status of NRT2.1 (using phospho-specific antibodies)

  • Investigate if specific transcription factors regulate the kinases or phosphatases that modify NRT transporters

• Systems-level analysis:

  • Combine transcriptomic data on nitrogen-responsive transcription factors with:

    • Proteomic data on NRT2.1/NRT3.1 levels

    • Post-translational modification status

    • Transport activity measurements

  • This multi-omics approach can reveal regulatory networks linking transcriptional control to protein function

For example, research has shown that the NIGT1/HRS1 transcription factors repress nitrogen starvation responses, including regulation of NRT2.4 . Combining ChIP-qPCR studies of these factors with protein-level analysis of NRT transporters could reveal how transcriptional regulation impacts the functional transport complex.

How do NRT3.1 antibodies from different plant species compare in terms of epitope recognition and experimental applications?

Comparative analysis of NRT3.1 antibodies across plant species reveals important considerations:

Key differences and considerations:

• Epitope selection:

  • Most antibodies target N-terminal regions, which tend to be less conserved across species

  • This provides specificity but limits cross-species applications

  • Sequence alignment of NRT3.1/NAR2.1 proteins across species can help identify both conserved and species-specific regions for antibody development

• Experimental applications:

  • Arabidopsis antibodies have been extensively validated for western blotting, complex analysis, and co-immunoprecipitation

  • Maize antibodies have been validated by mass spectrometry, confirming specific detection of ZmNRT3.1A

  • Rice antibodies have been used primarily for expression analysis in different tissues

• Molecular weight variations:

  • The detected molecular weight varies from ~21 kDa (maize) to ~25 kDa (Arabidopsis)

  • These differences likely reflect actual protein size differences rather than detection artifacts

  • Researchers should be aware of expected molecular weight when working with a new species

When working across species, researchers should consider generating new antibodies if existing ones don't cross-react, or target highly conserved epitopes if cross-species recognition is desired.

What methodological adaptations are necessary when studying NRT3.1-NRT2 interactions in different plant species?

When studying NRT3.1-NRT2 interactions across different plant species, several methodological adaptations are necessary:

• Antibody selection and validation:

  • Verify antibody specificity in your species of interest

  • If cross-reactivity is poor, develop new species-specific antibodies

  • For Arabidopsis, maize, and rice, specific antibodies have been described

• Extraction protocol optimization:

  • Adjust detergent types and concentrations based on membrane composition differences

  • For maize and rice, which have more robust cell walls than Arabidopsis, more aggressive grinding methods may be required

  • Buffer composition may need optimization for each species

• Complex analysis adaptations:

  • The NRT2.1-NRT3.1 complex in Arabidopsis appears at ~150 kDa on blue native gels

  • In other species, complex size may differ due to variations in protein size or stoichiometry

  • Perform preliminary gradient native PAGE to determine optimal separation conditions

• Functional correlation studies:

  • The contribution of different NRT2 family members to high-affinity nitrate uptake varies across species

  • In rice, OsNRT2.3a plays a significant role , while AtNRT2.1 is dominant in Arabidopsis

  • Design species-appropriate functional assays (15NO3- uptake parameters may need adjustment)

• Species-specific considerations:

  • Arabidopsis: Focus on AtNRT2.1-AtNRT3.1 as the primary complex; well-characterized antibodies are available

  • Maize: Consider the larger family of ZmNRT3 proteins (ZmNRT3.1A and ZmNRT3.1B); specific antibodies against ZmNRT3.1A have been validated

  • Rice: Investigate the relationship between OsNAR2.1 and multiple OsNRT2 family members (OsNRT2.1, OsNRT2.2, OsNRT2.3a/b, OsNRT2.4)