Recombinant Solanum lycopersicum 27 kDa cell wall protein

What is the structural composition of tomato cell wall proteins?

Tomato cell wall proteins exhibit complex domain structures that determine their functions. Analysis of tomato protein domains, as exemplified in PP2C protein studies, reveals that they typically contain specific catalytic domains alongside variable regions. Using MEGA-X software and the neighbor-joining method with 1000 bootstrap support, researchers have constructed phylogenetic trees to classify these proteins into distinct subfamilies .

Protein structure analysis shows that some tomato proteins contain multiple functional domains. For example, certain PP2C proteins (SlPP2C41, SlPP2C2, and SlPP2C27) contain both PP2C catalytic domains and protein kinase catalytic (PKc) domains, while others like SlPP2C70 have three PP2C domains . This structural diversity likely contributes to functional specialization within the cell wall proteome.

The exon-intron structure of genes encoding cell wall proteins provides additional insights into their evolution and regulation. Exons are typically represented by coding regions (shown as yellow boxes in analyses), while introns appear as non-coding intervening sequences (grey lines) . This genomic organization influences protein expression patterns and potentially their functional roles in the cell wall.

What approaches are used for expression and purification of recombinant tomato cell wall proteins?

Recombinant expression of tomato cell wall proteins, including the 27 kDa protein, requires optimization of expression systems and purification protocols. According to limited information available, the 27 kDa cell wall protein can be successfully expressed and purified as a recombinant protein, though specific methodological details are sparse .

For recombinant expression of tomato proteins, researchers typically amplify the full-length coding sequence from cDNA using gene-specific primers. This approach has been successfully demonstrated with other tomato proteins like SlSnRK1, where the complete coding sequence was amplified and subsequently verified through yeast two-hybrid systems . The choice of expression vector and host system significantly impacts protein yield and functionality.

Purification strategies should account for the specific properties of cell wall proteins, which often contain post-translational modifications and may form interactions with cell wall polysaccharides. Multiple purification steps may be necessary to achieve high purity while maintaining protein activity. The purified recombinant proteins can then be used for functional assays, structural studies, and interaction analyses.

How can researchers analyze cell wall protein localization in tomato tissues?

Immunocytochemical labeling combined with Confocal Laser Scanning Microscopy (CLSM) provides powerful insights into the spatial distribution of cell wall proteins. This approach has successfully revealed the localization patterns of various cell wall components including extensin (detected with LM1 antibody), xylan (LM11), rhamnogalacturonan (LM16), and homogalacturonan (LM19 and LM20) in tomato fruit tissues .

The localization patterns of cell wall proteins often change during developmental processes such as fruit ripening. For instance, microscopic studies have shown that low methyl-esterified homogalacturonan (LM19 epitopes) is primarily located in cellular corners and junctions during the Breaker stage but shifts to intercellular areas as fruits reach the Red Ripe stage . These dynamic changes reflect the functional roles of these proteins during cell wall remodeling.

For quantitative assessment of protein distribution, researchers can employ ELISA-based glycome profiling. This technique facilitates quantitative visualization of changes in cell wall components between different genetic backgrounds or developmental stages. The results are typically expressed as concentration of p-nitrophenol (PNP, mg/ml) obtained through alkaline phosphatase activity linked to secondary antibodies that recognize specific primary antibodies targeting cell wall components .

What expression patterns do cell wall proteins exhibit across different tomato tissues?

Cell wall proteins show differential expression patterns across various tomato tissues, reflecting their tissue-specific functions. Quantitative RT-PCR analyses of gene expression levels provide insights into these patterns. For example, studies of SlSnRK1, a protein kinase that may interact with cell wall proteins, revealed highest expression in flowers, intermediate levels in leaves, and lowest expression in roots and stems .

Expression patterns can also change in response to environmental stimuli or pathogen infection. Research has shown that SlSnRK1 transcript levels increase in tomato leaves infected with TYLCCNV/TYLCCNB compared to leaves infected with TYLCCNV alone at 3 days post-inoculation (DPI) . This suggests that certain regulatory proteins respond dynamically to biotic stresses, potentially modulating cell wall protein function during defense responses.

For the 27 kDa cell wall protein specifically, researchers would need to conduct tissue-specific expression analyses using techniques such as quantitative RT-PCR, western blotting, or immunohistochemistry to establish its expression patterns. Understanding these patterns would provide valuable insights into the protein's biological roles in different plant tissues and developmental stages.

How do cell wall proteins interact with polysaccharide components?

Cell wall proteins form complex interactions with various polysaccharides to maintain cell wall architecture. In vitro binding assays with glycome profiling demonstrate these interactions, as shown in studies of arabinogalactan proteins (AGPs) and their binding to cell wall polysaccharides . The table below illustrates the binding patterns observed between AGPs isolated from different tomato lines and various cell wall polysaccharides:

('+': high signal intensity; '±': low signal intensity; '−': no signal)

These binding patterns reveal that AGPs interact strongly with rhamnogalacturonan and cellulose, with binding intensities varying depending on the genetic background and ripening stage. For instance, AGPs from wild-type and RNAi#7 line show strong binding to cellulose, while those from overexpression lines (OEX#1 and OEX#2) exhibit weaker binding .

The APAP1 complex, a proteoglycan in which AGP binds to matrix polysaccharides, plays a significant role in these interactions. Studies with APAP1 mutants have shown increased levels of rhamnogalacturonan, homogalacturonan, and xylan, indicating that this complex affects the binding of these polymers to the cell wall .

How do genetic modifications of cell wall proteins affect fruit development and ripening?

Genetic modifications of genes encoding cell wall proteins significantly impact fruit development, particularly cell wall integrity during ripening. Studies using transgenic tomato lines with altered expression of SlP4H3 (a gene involved in protein hydroxylation) demonstrate these effects . The table below summarizes key phenotypic changes observed in these transgenic lines:

Microscopic visualization reveals that tissues from transgenic lines (OEX#1, OEX#2, and RNAi#7) show more damage in the Red Ripe stage compared to wild-type fruits . The cell walls in these transgenic lines exhibit disturbed continuity with visible interruptions and excessive swelling after ripening .

These structural changes correlate with alterations in cell wall component distribution. Overexpression of SlP4H3 affects extensin distribution, causing increased signal intensity in the cell wall and large conglomerates in cytoplasmic compartments that are absent in wild-type fruits . The transgenic lines also show a 20-30% increase in xylan and a 10% increase in rhamnogalacturonan, suggesting disruption of normal cell wall degradation during ripening .

What methodologies can detect protein-protein interactions involving cell wall proteins?

Several complementary methodologies can identify and characterize protein-protein interactions involving cell wall proteins. The yeast two-hybrid system provides a powerful approach for initial identification of interacting partners. This technique has been successfully employed to identify SlSnRK1 as an interacting partner for a viral protein in tomato, using a cDNA library fused to the GAL4 activation domain and the bait protein fused to the GAL4 DNA-binding domain .

For the 27 kDa cell wall protein, researchers could use:

• Yeast two-hybrid screening to identify potential interacting partners from a tomato cDNA library

• Co-immunoprecipitation followed by mass spectrometry to verify interactions in planta

• Bimolecular fluorescence complementation (BiFC) to visualize interactions in live cells

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to determine binding kinetics and affinities

Identified interactions can provide valuable insights into the functional roles of the 27 kDa cell wall protein within the complex network of cell wall components and regulatory machinery.

How do post-translational modifications affect cell wall protein function?

Post-translational modifications significantly influence cell wall protein function, affecting their activity, localization, and interactions. In tomato, hydroxylation of proline residues represents a critical modification that impacts protein structure and function. Studies with transgenic lines having altered expression of SlP4H3, a prolyl-4-hydroxylase gene, demonstrate that changes in this post-translational modification affect cell wall protein distribution and fruit development .

Phosphorylation represents another important post-translational modification. The presence of protein kinase catalytic domains in some tomato proteins, such as certain members of the PP2C family, suggests that phosphorylation plays a regulatory role . While the search results don't specifically address phosphorylation of the 27 kDa cell wall protein, SNF1-related kinases like SlSnRK1 may potentially target cell wall proteins as part of regulatory networks .

For comprehensive analysis of post-translational modifications in the 27 kDa cell wall protein, researchers could employ:

• Mass spectrometry to identify specific modification sites

• Site-directed mutagenesis to evaluate the functional significance of modified residues

• Phosphoproteomic approaches to characterize phosphorylation events

• In vitro enzyme assays to assess modification kinetics

These approaches would provide mechanistic insights into how post-translational modifications regulate the 27 kDa protein's function in the dynamic cell wall environment.

What bioinformatic approaches aid in predicting cell wall protein functions?

Sophisticated bioinformatic approaches enable researchers to predict cell wall protein functions based on sequence and structural features. Phylogenetic analysis using software like MEGA-X helps classify proteins into functional subfamilies, as demonstrated with the PP2C family in tomato where proteins were grouped into 12 subfamilies (A-L) .

Domain analysis identifies functional regions within proteins that indicate potential activities. For example, analysis of PP2C proteins revealed that alongside PP2C catalytic domains, some proteins (SlPP2C41, SlPP2C2, and SlPP2C27) also contain protein kinase catalytic domains . This combination of domains suggests dual functionality in signaling pathways.

Motif analysis identifies conserved sequences that may have specific functions. In the PP2C family, 19 motifs were identified, with motifs 1 and 2 present in almost all members, indicating their conserved functions . Other motifs appear subfamily-specific; for instance, motif 7 was found exclusively in subfamily D, while motifs 20 and 15 were restricted to subfamily C .

For the 27 kDa cell wall protein, researchers could apply these bioinformatic approaches to:

• Identify conserved domains and motifs indicating potential functions

• Predict three-dimensional structure using homology modeling

• Identify potential interaction partners through co-expression analysis

• Compare with homologous proteins across plant species to infer evolutionary conservation

These computational analyses would generate testable hypotheses about the protein's function that could guide experimental investigations.

How do cell wall proteins respond to biotic and abiotic stresses?

Cell wall proteins play critical roles in plant responses to biotic and abiotic stresses, undergoing changes in expression, localization, and activity. While the search results don't directly address the 27 kDa cell wall protein's stress responses, they provide insights into how tomato proteins respond to viral infections.

Quantitative RT-PCR analysis has shown that SlSnRK1 transcript levels increase in tomato leaves infected with TYLCCNV/TYLCCNB compared to leaves infected with TYLCCNV alone at 3 days post-inoculation . This suggests that certain regulatory proteins are upregulated in response to pathogen infection, potentially modulating downstream targets including cell wall proteins.

The cell wall undergoes significant remodeling during stress responses, involving changes in cell wall protein distribution. Immunocytochemical labeling studies can visualize these changes, as demonstrated for various cell wall components in tomato fruits . Similar approaches could be applied to study the 27 kDa cell wall protein's localization under different stress conditions.

For comprehensive analysis of the 27 kDa cell wall protein's stress responses, researchers could:

• Conduct transcriptomic and proteomic analyses under various stress conditions

• Perform immunolocalization studies to track protein redistribution during stress

• Analyze transgenic plants with altered expression levels under stress conditions

• Investigate potential post-translational modifications triggered by stress

These studies would elucidate the protein's role in stress adaptation mechanisms, potentially revealing applications for improving crop resilience.