Recombinant Solanum lycopersicum Histone H4

What is the genetic identity of Histone H4 in Solanum lycopersicum?

Histone H4 in Solanum lycopersicum is a protein-coding gene with Entrez Gene ID 544081. The gene produces a highly conserved nuclear protein that forms part of the nucleosome core structure. The corresponding mRNA sequence is documented under accession number NM_001247192.2, with the protein sequence available as NP_001234121.1 . The gene belongs to a small gene family in the tomato genome, with two cDNA variants that show 81% identity in their coding regions. Both variants are polyadenylated, suggesting post-transcriptional regulation mechanisms common to eukaryotic mRNAs .

How does the structure of Solanum lycopersicum Histone H4 compare with histone H4 proteins from other species?

Histone H4 is one of the most evolutionarily conserved proteins across eukaryotes. While specific comparative data for tomato H4 is limited in the provided sources, the high conservation suggests that tomato H4 likely maintains the structural characteristics essential for nucleosome assembly. The two identified tomato H4 cDNA clones were isolated using a heterologous probe from barley (Hordeum vulgare L.), indicating sufficient sequence similarity for cross-species hybridization . This conservation facilitates experimental approaches that can leverage protocols developed for histone H4 proteins from better-characterized species.

What is the expression pattern of Histone H4 in tomato tissues?

Histone H4 expression in tomato follows a tissue-specific and developmentally regulated pattern. The H4 message is abundant in young, actively dividing tissues and significantly reduced in older tissues . In the shoot apical meristem (SAM), H4 expression exhibits dynamic spatial regulation:

• In juvenile vegetative apex: H4 mRNA is detectable in both central and peripheral regions

• In mature vegetative meristem: H4-expressing cells localize to the peripheral zone, extending into provascular strands and rib meristem, while the central zone shows minimal expression

• After floral transition: H4 mRNA is expressed throughout the floral meristem

Notably, H4-expressing cells frequently occur in clusters, suggesting a partial synchronization of cell divisions in the shoot apex . This expression pattern correlates with tissues undergoing active cell division, consistent with histone H4's role in DNA packaging during replication.

What are the recommended methods for cloning and expressing recombinant Solanum lycopersicum Histone H4?

For cloning and expression of recombinant tomato histone H4, researchers should consider the following methodological approach:

• Source material selection: Use young, actively dividing tomato tissues (shoot tips, developing fruits) where H4 expression is highest .

• Cloning strategy:

  • The complete ORF sequence is 312bp in length

  • Standard vectors such as pcDNA3.1+/C-(K)DYK have been successfully used for histone H4 expression

  • When designing primers, consider the high GC content often found in histone genes

• Expression systems:

  • Bacterial expression (E. coli) provides high yield but lacks post-translational modifications

  • Insect cell systems may better preserve structural integrity and modifications

  • Plant-based expression systems can maintain species-specific modifications

• Purification approach:

  • Affinity tags (His, FLAG) facilitate purification but may affect function

  • Consider tag removal for functional studies

  • Acid extraction can be effective for histone isolation

When optimizing expression conditions, researchers should account for potential toxicity if overexpressed, as excess histone proteins can disrupt normal chromatin structure in host cells.

How can researchers verify the authenticity and activity of recombinant Solanum lycopersicum Histone H4?

Verification of recombinant tomato histone H4 should include multiple complementary approaches:

• Sequence verification: Confirm the nucleotide sequence matches NM_001247192.2 or other documented variants .

• Protein characterization:

  • SDS-PAGE should show a band at approximately 11 kDa

  • Mass spectrometry can confirm the exact molecular weight and sequence

  • Western blot using anti-histone H4 antibodies (cross-reactivity with plant histones should be verified)

• Functional verification:

  • Nucleosome assembly assays in vitro

  • DNA binding assays to confirm interaction with DNA

  • Incorporation into chromatin when added to nuclear extracts

• Post-translational modification analysis:

  • Mass spectrometry to identify acetylation, methylation, or other modifications

  • Modification-specific antibodies for Western blotting

  • The presence of specific modifications may indicate proper folding and recognition by modifying enzymes

For definitive confirmation, complementation studies in histone H4-depleted systems can demonstrate functional equivalence to native H4 proteins.

How does Solanum lycopersicum Histone H4 function in chromatin organization and gene regulation?

Tomato histone H4, like all H4 proteins, plays a fundamental role in chromatin organization by forming the nucleosome core particle together with H3, H2A, and H2B. The specific functions in tomato include:

• Developmental regulation: The dynamic expression pattern of H4 in the shoot apical meristem suggests a role in regulating developmental transitions. The shift in H4 expression domains correlates with changes in mitotic activity during vegetative growth and flowering transition .

• Cell cycle coordination: The clustering of H4-expressing cells suggests a role in coordinating cell division within specific domains of the meristem, potentially facilitating organized tissue development .

• Gene regulation through modifications: Histone H4 can be acetylated by histone acetyltransferases like GCN5, which is known to acetylate histones H3 and H4 in plants . These modifications alter chromatin accessibility and thereby regulate gene expression.

The presence of H4 throughout the floral meristem after floral transition indicates its importance in the gene expression changes required for reproductive development in tomato .

What is the relationship between histone H4 and other epigenetic factors in tomato?

Histone H4 functions within a complex epigenetic landscape in tomato that includes:

• Interaction with histone acetyltransferases: The SlGCN5 complex, which includes SlADA2a and SlADA2b proteins, can acetylate histones including H4 . This HAT activity is crucial for developmental processes in tomato.

• Coordination with other histone modifications: Tomato epigenome studies have revealed 26 different histone modifications that work together to establish distinct chromatin states . H4 modifications likely interact with modifications on other histones to create specific epigenetic environments.

• Role in genome topology: Histone modifications, including H3K9ac, have been shown to influence the three-dimensional organization of the tomato genome . As a core nucleosomal protein, H4 and its modifications contribute to these topological features.

• Developmental regulation: The SlGCN5 HAT complex regulates genes like SlWUS in the shoot apical meristem, suggesting that histone acetylation, potentially including H4 acetylation, is important for maintaining meristem function .

This intricate network of interactions positions histone H4 as a crucial component in establishing and maintaining epigenetic states that influence tomato development and gene expression.

What advanced techniques are employed for studying the interaction of recombinant histone H4 with other nuclear proteins in tomato?

Advanced researchers investigating tomato histone H4 interactions employ several sophisticated techniques:

• Chromatin Immunoprecipitation (ChIP):

  • ChIP-seq using anti-H4 or modification-specific antibodies identifies genomic regions associated with H4

  • ChIP followed by mass spectrometry (ChIP-MS) identifies proteins co-occurring with H4 in chromatin

• Proximity-based labeling techniques:

  • BioID or TurboID fused to H4 allows identification of proximal proteins in vivo

  • APEX2-based proximity labeling for rapid capture of transient interactions

• Fluorescence-based interaction studies:

  • Fluorescence Resonance Energy Transfer (FRET) to study direct protein-protein interactions

  • Bimolecular Fluorescence Complementation (BiFC) as demonstrated with SlGCN5 and SlADA2a/b proteins

  • Single-molecule tracking to observe H4 dynamics in living plant cells

• Protein complex analysis:

  • Co-immunoprecipitation followed by mass spectrometry

  • Size exclusion chromatography to identify native complexes containing H4

  • Cross-linking mass spectrometry (XL-MS) to map interaction interfaces

• In vitro reconstitution:

  • Nucleosome reconstitution assays with recombinant tomato histones

  • Single-molecule FRET to study conformational changes in reconstituted nucleosomes

These techniques enable researchers to build comprehensive interaction maps for histone H4 in the context of tomato nuclear architecture and gene regulation.

What are the known post-translational modifications of Solanum lycopersicum Histone H4 and their functional significance?

While the search results don't provide comprehensive data specifically on tomato H4 modifications, evidence from studies of the tomato epigenome and histone-modifying enzymes allows us to infer likely modifications and their functions:

• Acetylation: The presence of the SlGCN5 histone acetyltransferase complex in tomato indicates that H4 acetylation likely occurs, as GCN5 has been shown to acetylate H4 in other plant species . Functional significance includes:

  • Promotion of an open chromatin state for active transcription

  • Regulation of developmental processes, particularly in meristematic tissues

  • Potential role in defining genome topology and organization

• Methylation: The tomato epigenome study identified numerous histone methylation marks that likely include H4 methylation. These modifications typically:

  • Contribute to heterochromatin formation and gene silencing

  • Establish boundaries between active and inactive chromatin regions

  • Work in concert with other histone modifications to define chromatin states

• Other modifications: Based on conservation with other species, tomato H4 may also undergo phosphorylation, ubiquitination, and SUMOylation, each contributing to specific aspects of chromatin regulation.

The dynamic nature of these modifications allows for rapid response to developmental cues and environmental signals, as evidenced by the changing patterns of histone H4 expression during tomato shoot apex development .

How do researchers analyze histone H4 modifications in tomato chromatin?

Researchers employ multiple complementary techniques to analyze histone H4 modifications in tomato:

• Mass Spectrometry-based approaches:

  • Bottom-up proteomics for identification of specific modification sites

  • Top-down proteomics for analysis of combinatorial modifications

  • Quantitative MS to measure modification abundance across conditions

• Chromatin Immunoprecipitation (ChIP) techniques:

  • ChIP-seq using modification-specific antibodies maps genomic distribution

  • ChIP-qPCR for targeted analysis of specific genomic regions

  • Sequential ChIP to identify co-occurrence of different modifications

• Imaging techniques:

  • Immunofluorescence microscopy using modification-specific antibodies

  • Super-resolution microscopy to visualize spatial distribution in nucleus

  • Live-cell imaging with modification-specific probes for temporal dynamics

• Functional assays:

  • In vitro enzyme assays using recombinant modifying enzymes

  • Nucleosome reconstitution with modified histones

  • Mutation of modification sites followed by phenotypic analysis

The comprehensive epigenome study of tomato employed several of these techniques to characterize 26 different histone modifications and their relationship to genome topology , providing a framework for specific analysis of H4 modifications.

How can recombinant Solanum lycopersicum Histone H4 be used to study tomato development?

Recombinant tomato histone H4 provides a valuable tool for investigating developmental processes in tomato:

• Meristem development studies:

  • The changing pattern of H4 expression during shoot apex development suggests its importance in regulating cell division patterns

  • Tagged recombinant H4 can be used to track chromatin dynamics during meristem transitions

  • In vitro binding studies can identify developmental regulators that interact with H4-containing chromatin

• Chromatin reconstitution experiments:

  • Nucleosomes assembled with recombinant tomato H4 can be used in binding assays with developmental transcription factors

  • These studies can reveal how chromatin structure influences the accessibility of developmental gene promoters

• Epigenetic manipulation:

  • Overexpression of wild-type or mutant H4 (with modification sites altered) can reveal the importance of specific residues

  • Such studies can connect epigenetic mechanisms to developmental outcomes

  • The observation that H4-expressing cells occur in clusters suggests synchronized divisions that may be manipulated for developmental studies

• Reproductive development:

  • The distribution of H4 throughout floral meristems after transition indicates its role in reproductive development

  • Recombinant H4 can be used to study chromatin changes during flower development and fruit formation

These applications can provide insights into the molecular mechanisms underlying developmental transitions in tomato, potentially identifying targets for crop improvement.

What is the significance of histone H4 in understanding epigenetic regulation in crop plants?

Histone H4 serves as a critical component for understanding epigenetic regulation in tomato and other crop plants:

• Evolutionary conservation and divergence:

  • The high conservation of H4 across species allows comparative studies that reveal plant-specific adaptations

  • Understanding tomato-specific H4 modifications can highlight unique regulatory mechanisms in this important crop

• Developmental plasticity:

  • Histone modifications, including those on H4, contribute to developmental plasticity in response to environmental conditions

  • This plasticity is crucial for adaptation of crop plants to changing environments

• Integration with regulatory networks:

  • The interaction of H4 with the SlGCN5 complex and its role in regulating genes like SlWUS demonstrates how epigenetic mechanisms integrate with developmental regulatory networks

  • Such integration is fundamental to understanding complex traits in crop plants

• Genome topology and gene regulation:

  • Recent research has shown that histone modifications shape genome topology in tomato

  • This three-dimensional organization influences gene expression patterns relevant to agronomic traits

• Translational potential:

  • Understanding H4-mediated epigenetic regulation provides potential targets for breeding or biotechnological approaches to crop improvement

  • Engineered changes to H4 modification patterns could potentially influence traits like fruit development, stress tolerance, or yield

The detailed study of tomato histone H4 thus contributes to a broader understanding of epigenetic mechanisms that may be leveraged for crop improvement across multiple species.

What are common challenges in producing functional recombinant Solanum lycopersicum Histone H4?

Researchers working with recombinant tomato histone H4 frequently encounter several technical challenges:

• Expression system limitations:

  • Bacterial expression systems may yield incorrectly folded protein lacking essential post-translational modifications

  • Eukaryotic expression systems may introduce non-native modifications

  • The small size (approximately 11 kDa) of H4 can make it difficult to separate from contaminants

• Solubility issues:

  • Histones are highly basic proteins that tend to aggregate or precipitate during purification

  • Maintaining solubility often requires non-physiological conditions (high salt, detergents) that may affect functional studies

  • Refolding from inclusion bodies may be necessary but can reduce functional protein yield

• Modification heterogeneity:

  • Recombinant H4 may acquire different modification patterns depending on the expression system

  • These modifications can affect functional studies if not properly characterized

  • Achieving homogeneous modification states for specific studies is technically challenging

• Nucleosome assembly difficulties:

  • Reconstituting functional nucleosomes with recombinant histones requires careful optimization

  • Tomato-specific histone variants may have unique assembly requirements not addressed in standard protocols

  • The two identified H4 variants (81% identical in coding regions) may have distinct functional properties

• Validation challenges:

  • Limited availability of tomato-specific antibodies for histone H4 and its modifications

  • Cross-reactivity of antibodies with other plant histones must be carefully controlled

  • Functional validation often requires species-specific assays not widely established for tomato

Addressing these challenges requires careful optimization of expression systems, purification protocols, and validation methodologies specific to tomato histone proteins.

How can researchers resolve contradictory results in studies of histone H4 function in tomato?

When faced with contradictory results in tomato histone H4 studies, researchers should consider these methodological approaches:

• Biological context considerations:

  • The dynamic expression pattern of H4 across development stages means that results may differ based on tissue type and developmental stage examined

  • Different results may reflect genuine biological differences rather than experimental artifacts

  • Careful documentation of experimental conditions including plant age, tissue type, and growth conditions is essential

• Technical validation strategies:

  • Use multiple independent techniques to measure the same parameter

  • For example, combine ChIP-seq, in situ hybridization, and immunofluorescence to validate localization patterns

  • The observation that H4 expression changes during development requires precise staging of samples

• Genetic approaches:

  • Generate knockout/knockdown lines for H4 genes (challenging due to potential redundancy)

  • Use complementation experiments with variant forms to test specific hypotheses

  • Consider CRISPR-based approaches for precise modification of endogenous H4 genes

• Systematic analysis of variables:

  • Systematically test the effects of buffer conditions, protein concentrations, and experimental timing

  • For recombinant protein studies, compare different expression systems and purification methods

  • For in vivo studies, compare results across different tomato cultivars to identify genotype-specific effects

• Integration with broader epigenetic context:

  • Consider the interaction of H4 with the 26 histone modifications identified in tomato

  • Examine whether contradictory results might reflect different chromatin states or genome topological features

  • The relationship between H4 and histone acetyltransferases like SlGCN5 may help resolve apparently conflicting observations

By systematically addressing these factors, researchers can reconcile contradictory results and develop a more comprehensive understanding of histone H4 function in tomato.