Recombinant Nicotiana tabacum Actin-104

What are the optimal storage conditions for recombinant Nicotiana tabacum Actin-104?

For optimal stability and activity retention of recombinant Nicotiana tabacum Actin-104, storage at -20°C is recommended for routine use. For extended storage periods, conservation at -80°C is advised to minimize protein degradation. The shelf life varies by formulation: liquid preparations maintain integrity for approximately 6 months at these temperatures, while lyophilized forms remain stable for up to 12 months .

It is crucial to avoid repeated freeze-thaw cycles, which significantly compromise protein quality. Working aliquots can be maintained at 4°C for up to one week without substantial loss of activity. When preparing the protein for experimental use, brief centrifugation of the vial is recommended prior to opening to ensure all content is collected at the bottom .

How should recombinant Nicotiana tabacum Actin-104 be reconstituted for experimental use?

For optimal reconstitution of recombinant Nicotiana tabacum Actin-104, researchers should follow this methodological approach:

• Use deionized sterile water as the reconstitution medium

• Prepare to a final concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being standard practice)

• Prepare smaller working aliquots to minimize freeze-thaw cycles

• Store reconstituted aliquots at -20°C or -80°C for long-term storage

This reconstitution protocol ensures protein stability while maintaining functional properties needed for downstream applications. The addition of glycerol serves as a cryoprotectant, preventing ice crystal formation that could denature the protein structure during freeze-thaw cycles .

What expression systems are most effective for producing recombinant Nicotiana tabacum Actin-104?

While commercially available recombinant Nicotiana tabacum Actin-104 is typically produced in mammalian cell expression systems , research indicates several viable plant-based expression platforms may offer advantages for actin protein production.

The choice of expression system significantly impacts yield and functionality:

• Mammalian cell systems: Provide proper folding and post-translational modifications, with yields typically achieving >85% purity as determined by SDS-PAGE .

• Plant-based systems: Several Nicotiana platforms show promise:

  • Nicotiana benthamiana transient expression: Allows rapid protein production within 4-5 days post-infiltration

  • Nicotiana tabacum BY-2 cells: Provide controlled growth conditions in suspension culture

  • Plant-cell-pack (PCP) platform: Utilizes tobacco cells in a compact format for protein expression

• Cell-free expression: Particularly suitable for proteins containing intrinsically disordered regions (IDRs), using tobacco cell lysates

When comparing expression systems, researchers should consider that transient expression in N. benthamiana leaves with p19 silencing inhibitor co-expression has demonstrated up to 15-fold increases in recombinant protein yields .

How does p19 silencing inhibitor affect recombinant protein expression in Nicotiana systems?

The p19 silencing inhibitor from Tomato bushy stunt virus significantly enhances recombinant protein expression in Nicotiana systems through several mechanisms:

• Quantitative impact: Co-expression of p19 with recombinant proteins in N. benthamiana increases expression levels approximately 15-fold compared to expression without p19 .

• Expression vector dependency: The enhancement effect varies based on the genetic elements used. When trastuzumab antibody was expressed using different vector systems (numbered 102-106), p19 increased antibody concentration to ~2.3% of total soluble protein (TSP) when co-expressed with vector 103, but showed minimal effect with vectors 102 and 106 .

• Cultivar compatibility: When expressing recombinant proteins with p19, cultivar selection is critical. Studies show that while p19 enhances expression in some N. tabacum cultivars, others (except cultivar Little Crittenden) exhibit marked discoloration of infiltrated leaf areas and decreased protein expression .

• Temporal dynamics: Optimal expression levels are typically observed at 4-5 days post-infiltration when using p19, with diminishing returns at 6 days post-infiltration due to potential tissue necrosis .

These findings suggest that researchers should carefully optimize p19 co-expression conditions, including construct ratios, cultivar selection, and harvest timing to maximize recombinant actin production while minimizing adverse effects on plant tissue .

What subcellular targeting strategies optimize recombinant actin expression in plant systems?

Subcellular targeting significantly impacts both the yield and functionality of recombinant proteins in plant expression systems, including actin. Based on experimental evidence, the following targeting strategies show differential effects:

• Apoplast targeting: Secretion of recombinant proteins to the apoplast has demonstrated approximately 1.3-fold higher expression levels compared to cytosolic or ER retention, although these differences were not statistically significant (two-sided t-test; df = 9, α = 0.05) .

• ER retention: The endoplasmic reticulum provides benefits through molecular chaperones and limited protease activity, potentially enhancing protein stability.

• Cytosolic expression: While convenient for native actin folding, cytosolic expression of actin may interfere with endogenous actin filaments, potentially disrupting cellular integrity and intracellular trafficking .

• Plastid targeting: No detectable expression was observed for plastid-targeted recombinant proteins in N. benthamiana leaves, making this an unsuitable location for actin expression .

When expressing actin specifically, researchers should consider that cytosolic expression may lead to detrimental interactions with endogenous actin networks. Evidence suggests that when recombinant PHACTR1 (a phosphatase and actin regulator) was expressed in plant cells, it potentially interfered with intracellular actin filaments, causing loss of cell integrity. This effect was less pronounced when the protein was targeted to the ER or apoplast, suggesting that spatial isolation from cytosolic interaction partners may reduce negative effects .

How is Nicotiana tabacum Actin-104 related to other plant actin genes?

Nicotiana tabacum contains a complex actin gene family estimated to include between 20-30 actin genes based on Southern hybridization and library screening techniques, though the total gene family may be larger . Through genomic analysis and sequence comparison, tobacco actin genes demonstrate specific phylogenetic relationships with other plant actins:

• Evolutionary relationships: Nicotiana tabacum actin genes show varying degrees of homology to other plant actins. For example, the tobacco actin gene Tac25 demonstrates close phylogenetic relationship to the allelic potato actin genes Pac58 and Pac85, while another tobacco actin gene, Tac9, shows greater homology to potato actin Pac79 .

• Expression patterns: Different actin genes within N. tabacum exhibit tissue-specific expression patterns. Northern hybridization analysis revealed that Tac9 transcripts were detected across multiple tissues including root, leaf, stigma, and pollen, while Tac25 transcripts were exclusively detected in pollen RNA .

• Structural conservation: Tobacco actin genes demonstrate the typical exon-intron structure of plant actins, with Tac9 showing homology to the third exon of both Tac25 and the soybean actin gene Sac3 .

These relationships indicate that Actin-104 likely belongs to a specific subclade within the larger plant actin family, potentially sharing functional and structural similarities with other Solanaceae actin genes. The tissue-specific expression patterns suggest specialized functional roles for different actin isoforms within the plant.

What functional domains characterize Nicotiana tabacum Actin-104 and how do they compare to actins from other species?

Nicotiana tabacum Actin-104 contains several highly conserved functional domains characteristic of the actin protein family, which are essential for its structural and functional properties:

• ATP-binding domain: Located within the N-terminal region of the protein, this domain (identifiable in the sequence "AGFAGDDAPR AVFPSIVGRP") is critical for ATP hydrolysis, which powers actin polymerization and dynamics .

• Divalent cation binding sites: These sites bind Ca²⁺ or Mg²⁺, which are essential cofactors for actin function and stability.

• Hydrophobic binding pocket: This region facilitates actin-actin interactions during filament formation and interactions with actin-binding proteins.

• DNase I binding loop: A flexible region involved in interactions with various actin-binding proteins.

Comparative analysis reveals that Nicotiana tabacum Actin-104's sequence is highly conserved compared to actins from other plant species, particularly within the Solanaceae family. The high degree of sequence conservation (typically >90% identity in the coding regions) reflects the fundamental importance of actin's structural role across plant species.

Table 1: Key Functional Domains in Nicotiana tabacum Actin-104

This domain architecture enables Actin-104 to participate in critical cellular functions including cytoskeletal organization, cellular trafficking, and structural support in Nicotiana tabacum cells.

What are the optimal buffer conditions for maintaining Nicotiana tabacum Actin-104 stability in vitro?

Maintaining the stability and functionality of Nicotiana tabacum Actin-104 in vitro requires careful buffer optimization. Based on established protocols for plant actins, the following buffer conditions are recommended:

• Storage buffer composition:

  • 5 mM Tris-HCl (pH 8.0)

  • 0.2 mM CaCl₂

  • 0.2 mM ATP

  • 0.5 mM DTT (to prevent oxidation of cysteine residues)

  • 0.1 mM PMSF (protease inhibitor)

  • 5% (w/v) sucrose or 10-50% glycerol as cryoprotectant

• Experimental buffer for polymerization studies:

  • 10 mM Imidazole (pH 7.0)

  • 50 mM KCl

  • 1 mM MgCl₂

  • 1 mM EGTA

  • 0.2 mM ATP

  • 0.5 mM DTT

• Critical considerations:

  • Maintain pH between 7.0-8.0 to prevent denaturation

  • Include divalent cations (Ca²⁺ or Mg²⁺) for structural stability

  • Add fresh DTT before experiments to maintain reducing conditions

  • Keep ATP in buffers to prevent spontaneous depolymerization

  • Work at temperatures between 4-22°C to prevent protein degradation

For long-term storage, adding glycerol to a final concentration of 50% and storing at -20°C or -80°C is recommended. Avoid repeated freeze-thaw cycles by preparing small working aliquots that can be maintained at 4°C for up to one week .

How can researchers effectively analyze interactions between Nicotiana tabacum Actin-104 and actin-binding proteins?

Investigating interactions between Nicotiana tabacum Actin-104 and actin-binding proteins requires specialized techniques that preserve the native conformation and binding properties of both interaction partners. Several methodological approaches can be employed:

• Co-sedimentation assays:

  • Prepare F-actin by polymerizing G-actin in polymerization buffer

  • Incubate F-actin with purified actin-binding protein of interest

  • Ultracentrifuge at ~100,000 × g for 30 minutes

  • Analyze supernatant and pellet fractions by SDS-PAGE

  • Quantify binding by densitometry of protein bands

• Fluorescence-based approaches:

  • Label actin with pyrene at cysteine-374

  • Monitor polymerization kinetics by measuring fluorescence increase

  • Assess effects of binding proteins on polymerization rate and critical concentration

  • Alternatively, use FRET with appropriately labeled actin and binding proteins

• Surface Plasmon Resonance (SPR):

  • Immobilize either actin or binding protein on sensor chip

  • Flow partner protein at various concentrations

  • Measure association and dissociation kinetics

  • Calculate binding constants (KD, kon, koff)

• Microscopy techniques:

  • Visualize actin filaments using fluorescently labeled phalloidin

  • Perform total internal reflection fluorescence (TIRF) microscopy to observe single filament dynamics

  • Assess effects of binding proteins on filament structure, branching, or bundling

• Plant-specific considerations:

  • Plant actin-binding proteins may have different pH or salt optima than animal counterparts

  • Include appropriate plant cofactors that might be required for native interactions

  • Consider using plant-derived expression systems to ensure proper post-translational modifications

When studying the interactions between Nicotiana tabacum Actin-104 and phosphatase and actin regulator proteins (like PHACTR1), researchers should be aware that these interactions may interfere with intracellular actin filaments, potentially causing loss of cell integrity and inhibiting intracellular vesicle trafficking .

What imaging techniques are most effective for visualizing Nicotiana tabacum Actin-104 dynamics in plant cells?

Visualizing actin dynamics in plant cells presents unique challenges due to cell wall barriers, vacuolar compartmentalization, and autofluorescence. For studying Nicotiana tabacum Actin-104 dynamics in vivo, several advanced imaging approaches offer complementary insights:

• Fluorescent protein fusions:

  • Generate GFP/YFP/mCherry fusions with Actin-104

  • Express under native or tissue-specific promoters

  • Use transient expression via Agrobacterium infiltration in N. benthamiana

  • Consider subcellular targeting to minimize interference with endogenous actin

• Lifeact or ABD2-GFP reporters:

  • Express fluorescent reporters fused to actin-binding domains

  • Advantages: Minimal interference with actin dynamics

  • Limitations: May preferentially bind certain actin populations

  • Optimize expression levels to prevent artifacts

• Advanced microscopy techniques:

  • Spinning disk confocal microscopy: For rapid acquisition of dynamic processes

  • Variable angle epifluorescence microscopy (VAEM): For near-TIRF imaging of cortical actin

  • Super-resolution techniques (PALM/STORM): For nanoscale organization of actin filaments

  • Fluorescence recovery after photobleaching (FRAP): For measuring actin turnover rates

• Sample preparation considerations:

  • Use infiltration with latrunculin B or cytochalasin D as positive controls

  • Mount samples in appropriate buffers to maintain cell viability

  • Utilize vacuum infiltration for equal distribution of fixatives or probes

  • Consider physiological state and developmental stage of tissue

• Quantification approaches:

  • FilamentTracker or OpenTracker plugins for ImageJ/Fiji

  • Measure parameters including filament length, bundling, orientation, and density

  • Apply skeletonization algorithms for consistent analysis

  • Use kymographs to analyze filament dynamics over time

When interpreting results, researchers should be aware that expression of recombinant actins or actin regulators can potentially disrupt endogenous actin networks. In studies with PHACTR1, researchers observed that cytosolic expression led to browning and shrinking of plant cell packs, indicating potential necrosis, while targeting to ER or apoplast reduced these effects .

How does Nicotiana tabacum Actin-104 interact with plant-specific actin-binding proteins compared to other actin isoforms?

The interactions between Nicotiana tabacum Actin-104 and plant-specific actin-binding proteins (ABPs) reveal important functional distinctions from other actin isoforms. These interactions are characterized by:

• Isoform-specific binding profiles: Plant actins, including Nicotiana tabacum Actin-104, exhibit differential affinities for various ABPs compared to animal actins. These differences are often subtle but functionally significant, affecting filament dynamics and organization.

• Plant-specific ABP interactions: N. tabacum Actin-104 interacts with several classes of plant-specific ABPs, including:

  • Villins/Gelsolins: Regulate filament severing and capping

  • Formins: Nucleate and elongate actin filaments

  • Actin-depolymerizing factors (ADFs): Control filament turnover

  • Fimbrins: Crosslink actin filaments

• Regulatory mechanisms: The interactions between N. tabacum Actin-104 and ABPs are regulated by:

  • Calcium concentration: Affecting binding of Ca²⁺-sensitive ABPs

  • pH: Modulating ADF/cofilin activity

  • Phosphorylation: Altering binding affinities and activities

  • Redox state: Affecting interactions with redox-sensitive ABPs

• Functional implications: These specialized interactions contribute to actin's diverse roles in:

  • Cytoplasmic streaming

  • Cell division and expansion

  • Response to biotic and abiotic stresses

  • Organelle positioning and movement

The distinct interactions between Nicotiana tabacum Actin-104 and plant ABPs likely contribute to the specific cytoskeletal arrangements observed in tobacco cells. Research suggests that when recombinant actin-interacting proteins (such as PHACTR1) are expressed in plant cells, they may interfere with endogenous actin networks, disrupting cellular integrity and function .

What roles do Nicotiana tabacum actin genes play in response to pathogen infection and stress conditions?

Nicotiana tabacum actin genes, including Actin-104, play crucial roles in the plant's response to pathogen infection and various stress conditions through dynamic cytoskeletal rearrangements and signaling pathway modulation:

• Pathogen response mechanisms:

  • Actin cytoskeleton reorganization occurs during pathogen attack

  • Actin filaments guide secretory vesicles to infection sites

  • Actin-dependent papilla formation creates physical barriers against pathogen penetration

  • Actin dynamics influence programmed cell death during hypersensitive response

• Silencing suppressor interactions:

  • Plant viral suppressors like p19 influence actin-dependent RNA silencing pathways

  • P19 from Tomato bushy stunt virus enhances recombinant protein expression by suppressing RNA silencing

  • This silencing suppression affects host defense responses by altering RNA-dependent RNA polymerase (RDR) activities

• Stress response pathways:

  • Abiotic stresses trigger rapid actin cytoskeleton remodeling

  • Actin genes show differential expression patterns under various stress conditions

  • Actin-binding proteins modulate filament dynamics during stress adaptation

  • RNA-dependent RNA polymerase 1 (RDR1) from N. tabacum, which is induced by salicylic acid, contributes to antiviral defense

• Transcriptional regulation:

  • Different actin isoforms show tissue-specific expression patterns

  • Tac9 transcripts are detected across multiple tissues, while Tac25 transcripts are found exclusively in pollen

  • Stress conditions may alter the expression patterns of specific actin isoforms

  • ARF transcription factors (NtARF7 and NtARF19) are upregulated in transgenic N. tabacum plants, potentially influencing actin gene expression in response to stress

Understanding these complex interactions is crucial for developing strategies to enhance plant resistance to pathogens and environmental stresses, and may inform approaches for optimizing recombinant protein expression in plant systems.

How do mutations in the Nicotiana tabacum actin gene family affect plant development and morphology?

Mutations in the Nicotiana tabacum actin gene family have profound effects on plant development and morphology due to actin's central role in numerous cellular processes. These effects manifest in multiple developmental pathways:

• Root development alterations:

  • Actin mutations can disrupt root hair formation and elongation

  • Root architecture may be compromised due to altered cell division planes

  • Auxin transport patterns may be disrupted, affecting root gravitropism

  • NtARF7 and NtARF19 upregulation in transgenic N. tabacum plants suggests involvement in root-related biological processes

• Cell division and expansion defects:

  • Actin is essential for cytokinesis and phragmoplast formation

  • Mutations can lead to incomplete cell divisions and multinucleated cells

  • Cell expansion may be compromised due to altered vesicle trafficking

  • Trichome development and branching patterns may be affected

• Reproductive development impacts:

  • Pollen-specific expression of certain actin isoforms (e.g., Tac25) suggests critical roles in reproduction

  • Pollen tube growth, which relies heavily on actin dynamics, may be compromised

  • Embryo sac development can be affected, reducing female fertility

  • Seed set and development may be reduced in actin mutants

• Vascular tissue organization:

  • Actin cytoskeleton guides the deposition of cell wall materials

  • Vascular tissue patterning may be disrupted in actin mutants

  • Water and nutrient transport efficiency may be compromised

  • Secondary cell wall formation may be altered, affecting stem strength

• Hormone response modifications:

  • Actin dynamics influence hormone receptor trafficking and recycling

  • Auxin transport and signaling are particularly dependent on actin

  • Rolb-transgenic N. tabacum plants show upregulation of NtARF7 and NtARF19, suggesting altered auxin signaling networks

  • Hormone-induced morphological changes may be attenuated in actin mutants

The complexity of the actin gene family in N. tabacum, with an estimated 20-30 genes , suggests functional redundancy but also specialization. This makes it challenging to predict the precise effects of mutations in individual actin genes, as compensation by other family members may occur to varying degrees depending on expression patterns and functional overlap.