Recombinant Solanum lycopersicum Ornithine decarboxylase (ODC)

What is the genetic structure of Ornithine Decarboxylase in Solanum lycopersicum?

Ornithine decarboxylase in tomato (Solanum lycopersicum) is represented by a single-copy gene as confirmed by Southern-blot analysis and copy-number reconstructions. When genomic DNA digested with various restriction enzymes (DraI, EcoRV, AccI, HincII, and XbaI) was hybridized to the entire tomato ODC cDNA, specific hybridization patterns were observed. The ODC cDNA contains one recognition site for AccI, HincII, and XbaI, but none for EcoRV and DraI. The probe hybridized to two AccI fragments, two HincII fragments, and two XbaI fragments, while only one hybridizing EcoRV fragment and one hybridizing DraI fragment were detected, even under low stringency washing conditions .

What is the primary function of Ornithine Decarboxylase in tomato plants?

Ornithine Decarboxylase (ODC) catalyzes the synthesis of putrescine from ornithine, which is the initial key step in the polyamine (PA) biosynthetic pathway in eukaryotic cells. In plants, polyamines (putrescine, spermidine, and spermine) are small molecules, charged at physiological pH, that have been implicated in a wide range of growth and developmental processes, including floral and fruit development, as well as responses to senescence and stress conditions. ODC has been described as the enzyme controlling polyamine biosynthesis in tomato and other plant species .

How is ODC gene expression regulated during tomato fruit development?

The ODC gene in tomato has been shown to be up-regulated during early fruit growth. Research demonstrates that ODC expression is transiently increased by plant hormones, specifically 2,4-dichlorophenoxyacetic acid and gibberellic acid, which induce fruit development. This regulation suggests ODC plays a significant role in the initial stages of fruit development, potentially through modulation of polyamine levels that influence cell division and expansion processes .

What methods are effective for cloning tomato ODC cDNA?

For successful cloning of tomato ODC cDNA, researchers have employed RT-PCR using primers corresponding to conserved regions of ODC. A proven approach involves:

• First-strand cDNA synthesis using poly(A+) RNA from unpollinated tomato ovaries

• PCR amplification using primers corresponding to positions 97-116 (ODC-5′) and 638-659 (ODC-3′) derived from a partial ODC cDNA sequence from tobacco

• Cloning of the amplified fragment (approximately 564-bp) into a suitable vector (e.g., pGEM-T)

• Sequence confirmation of the cloned fragment

• Construction of a cDNA library using poly(A+) RNA from unpollinated ovaries

• Screening of plaque-forming units using the RT-PCR fragment as a probe to isolate full-length cDNAs

This methodology has successfully yielded functional ODC cDNA clones from tomato .

What expression systems are optimal for producing active recombinant Solanum lycopersicum ODC?

While the search results don't specifically address expression systems for tomato ODC, effective approaches for recombinant expression of plant decarboxylases can be inferred from related research. For ancestral decarboxylases, researchers have successfully used Escherichia coli BL21 as an expression host. After expression, proteins are purified and their decarboxylase activity can be assayed by detection of CO2 release at appropriate temperature and pH conditions .

For tomato ODC specifically, bacterial expression systems like E. coli are commonly employed for initial characterization, while yeast systems (Saccharomyces cerevisiae or Pichia pastoris) may provide more appropriate post-translational modifications. Expression vectors containing strong promoters and appropriate tags for purification (His-tag, GST-tag) facilitate isolation of the recombinant protein while maintaining enzymatic activity.

What are the kinetic parameters of recombinant Solanum lycopersicum ODC?

While the specific kinetic parameters for Solanum lycopersicum ODC are not detailed in the provided search results, comparative data from related ODCs can provide insight. For example, the Fusobacterium necrophorum ODC demonstrates the following kinetic parameters:

This data demonstrates that ODC typically has highest specificity for ornithine (as measured by kcat/Km), with significantly lower efficiency for alternative substrates like arginine and lysine . When characterizing recombinant tomato ODC, researchers should examine similar parameters to determine substrate preference and catalytic efficiency.

How does substrate specificity of tomato ODC compare to ODCs from other organisms?

ODCs from different organisms exhibit varying substrate specificities. While tomato ODC is primarily specific for L-ornithine, research on ancestral ODCs reveals interesting comparative insights. For instance, F. necrophorum ODC shows a strong preference for L-ornithine with a kcat/Km 313-fold greater than with L-arginine and 138-fold greater than with L-lysine. This substrate preference is manifested by a lower Km and higher turnover rate for L-ornithine compared to L-arginine or L-lysine .

In contrast, some ancestral decarboxylases like the bifunctional ODC/LDC from Thermoanaerobacterium thermosaccharolyticum exhibit equal preference for L-ornithine and L-lysine, with only 2.2-fold less preference for L-arginine. When comparing enzyme efficiencies, the T. thermosaccharolyticum enzyme is approximately 3.7-fold less efficient with L-ornithine compared to the Clostridium botulinum ODC, primarily due to a lower turnover rate .

Understanding these comparative differences in substrate specificity is crucial when working with recombinant tomato ODC, as it provides context for evolutionary and functional relationships between plant and microbial decarboxylases.

How does ODC activity relate to polyamine biosynthesis in stress response pathways of tomato?

ODC catalyzes the conversion of ornithine to putrescine, which is the first committed step in polyamine biosynthesis. Polyamines have been implicated in stress responses in plants, including tomato. While the search results don't directly address ODC's role in stress response, research on wild tomato relatives suggests connections between polyamine biosynthesis and stress tolerance.

For example, in Solanum habrochaites, a wild relative of cultivated tomato with enhanced cold tolerance, jasmonate signaling plays an essential role in stress response. Under cold stress conditions (4°C), increased expression of genes involved in hormone signal transduction pathways was observed. The rootstock of S. habrochaites can enhance cold tolerance in grafted S. lycopersicum scions through jasmonate signaling . Since polyamines interact with various hormone signaling pathways, ODC activity may be modulated during stress responses to adjust polyamine levels, contributing to stress tolerance mechanisms.

What is the relationship between ODC activity and fruit development in tomato?

ODC gene expression is upregulated during early fruit growth induced by 2,4-dichlorophenoxyacetic acid and gibberellic acid in tomato . This suggests a critical role for ODC in the initial stages of fruit development, likely through its contribution to polyamine biosynthesis. Polyamines (putrescine, spermidine, and spermine) have been implicated in a wide range of growth and developmental processes, including floral and fruit development .

The transient increase in ODC expression during early fruit development indicates that polyamine biosynthesis may be particularly important during cell division and expansion phases. Polyamines can influence DNA synthesis, cell division, and membrane stability, all of which are essential processes during fruit set and early development. For researchers investigating fruit development in tomato, modulation of ODC activity through genetic approaches (overexpression, silencing) could provide valuable insights into the specific roles of polyamines at different developmental stages.

What are the optimal assay conditions for measuring recombinant tomato ODC activity?

Based on protocols used for related decarboxylases, the following assay conditions are recommended for measuring recombinant tomato ODC activity:

• Purify the recombinant enzyme using appropriate chromatography techniques

• Measure decarboxylase activity by detection of CO2 release

• Conduct assays at moderate temperature (e.g., 26-27°C) and neutral to slightly alkaline pH (approximately pH 7.7)

• Use L-ornithine as the primary substrate, with L-arginine and L-lysine as alternative substrates to confirm specificity

• Determine enzyme kinetic parameters (Km, kcat, kcat/Km) for each substrate

• Perform assays in triplicate to ensure reliability of results

This approach allows for accurate determination of enzyme activity and substrate preference . Additionally, researchers should consider including appropriate controls and enzyme inhibitors to validate assay specificity.

How can gene editing techniques be utilized to study ODC function in tomato plants?

Modern gene editing techniques provide powerful tools for studying ODC function in tomato plants. Researchers can employ CRISPR/Cas9 systems to:

• Create knockout mutants by introducing frameshift mutations in the ODC coding sequence

• Generate knockdown lines using CRISPR interference (CRISPRi) for partial reduction of expression

• Develop promoter modifications to alter expression patterns temporally or spatially

• Introduce specific amino acid substitutions to study structure-function relationships

When designing gene editing experiments, researchers should consider the single-copy nature of the ODC gene in tomato , which simplifies editing approaches but may complicate the generation of viable plants if ODC function is essential. Alternatively, RNAi-based approaches can be used to reduce ODC expression in specific tissues or developmental stages.

For functional validation, transformed plants should be analyzed for changes in polyamine levels, developmental phenotypes, and stress responses. Complementation studies with the wild-type gene or specific variants can confirm that observed phenotypes are due to altered ODC function rather than off-target effects.

How does ODC activity influence pest resistance in tomato?

Research on whitefly resistance in tomato provides indirect evidence connecting polyamine biosynthesis (which involves ODC) to pest resistance mechanisms. Studies of Solanum habrochaites sp. glabratum × S. lycopersicum populations identified two QTL regions in chromosomes 5 and 11 that positively correlated with whitefly oviposition and colocalized with a newly reported candidate susceptibility factor involved in polyamine biosynthesis .

Specifically, QTL11 includes a region with Acylsugar Acyl Transferase 3 (ASAT3), a gene associated with increased adult whitefly mortality, increased density of type IV trichomes, and higher abundance of acylsugars. Interestingly, an isoform of ASAT3 found in S. habrochaites sp. glabratum was hypothesized to be regiospecific in the transfer of acyl-groups to different positions than those previously characterized in S. lycopersicum .

Since polyamines produced via the ODC pathway can influence various metabolic processes, modulation of ODC activity might affect the production of defensive compounds or structures (like trichomes) that contribute to pest resistance. Researchers investigating ODC's role in pest resistance should consider how polyamine levels influence secondary metabolite production and defensive structures in tomato.

What approaches can be used to modulate ODC expression to enhance stress tolerance in tomato?

Several approaches can be employed to modulate ODC expression for enhanced stress tolerance:

• Overexpression strategies: Introducing additional copies of the native ODC gene or heterologous ODC genes under control of constitutive or stress-inducible promoters

• Grafting approaches: Using rootstocks with altered ODC expression to influence scion responses, similar to the approach used with S. habrochaites rootstocks that enhanced cold tolerance in S. lycopersicum scions

• Chemical modulation: Application of ODC inhibitors or polyamine precursors at specific developmental stages to alter polyamine metabolism

• Targeted promoter modifications: Using gene editing to modify the ODC promoter region to enhance responsiveness to specific stress conditions

• Co-expression with related enzymes: Coordinated modulation of multiple enzymes in the polyamine biosynthetic pathway to prevent bottlenecks

When implementing these approaches, researchers should carefully assess polyamine levels, expression of related genes, and physiological responses to stress conditions. The experimental design should include appropriate controls and consider potential pleiotropic effects of altered polyamine metabolism on growth, development, and yield.