The full-length nucleotide sequence of the class III WRKY TF gene ScWRKY5 (945 bp) was retrieved from the NCBI GenBank database under accession number MT003230.1.¹⁷

To enhance expression efficiency in Solanum lycopersicum, codon optimization of ScWRKY5 was performed using the GenScript Rare Codon Analysis Tool and the Optimizer Tool (http://genomes.urv.es/OPTIMIZER).^22,23 Parameters used:

After optimization, the CAI improved from 0.68 to 0.94, and GC content was reduced from 63.49% to 52.37%.^24–26 (Figure 2 and Table 1).

The optimized mRNA sequence was deduced using the Biomodel Transcription and Translation Tool (https://biomodel.uah.es/en/lab/cybertory/analysis/trans.htm) and provided in Supplementary Data 5.

Primers were designed using Primer3Plus, IDT PrimerQuest, and Geneious Prime.²² Parameters considered:

The optimized ScWRKY5 gene was inserted in silico into the pCAMBIA1302 vector between BamHI and XbaI using SnapGene software (Figures 3 and 4).^27,28 The final product of in silico PCR amplification of the 945 bp ScWRKY5 is depicted in Figure 7.

pCAMBIA1302 was selected primarily for its constitutive CaMV 35S promoter, ensuring strong, reliable transgene expression in dicotyledonous plants like tomato, along with its integrated mgfp5 reporter and hygromycin resistance marker for efficient selection and expression validation.

pCAMBIA1302 was selected for its features:

mRNA secondary structure was predicted using RNAfold v2.6.3. The minimum free energy (MFE) was −412.70 kcal/mol, indicating stability.²²

The optimized sequence was translated using ExPASy Translate.²² Physicochemical properties were analyzed via ProtParam (Table 3).²⁵

ENc and RSCU values were calculated using CodonW v1.4.4 and visualized as heat maps to assess codon usage bias reduction before and after optimization.

The following software tools were used throughout the study for various stages of sequence analysis, optimization, and validation (Table 4).

The steps undertaken in this research are summarized in the following flowchart in Figure 5, showing the overall methodology used in this study.

The ScWRKY5 gene (945 bp) encodes a 314-aa protein. Codon optimization:

In silico PCR confirmed specific amplification of the 945 bp product. Primers were validated for melting temperature, GC content, and restriction compatibility (Table 2) (See Figures 4 and 7).

Insertion of the optimized gene was confirmed by restriction mapping and sequencing simulation. ORF and promoter integrity were intact (Figure 3).

The translated ScWRKY5 protein showed:

The circular map represents the recombinant pCAMBIA1302::ScWRKY5 construct generated using SnapGene. Key vector features are annotated, including the CaMV 35S promoter, ORF of the codon-optimized ScWRKY5 gene, and the NOS terminator. Restriction sites (EcoRV, Aal) flank the insertion region for validation. The lower schematic confirms the proper translation frame, indicating a continuous ORF without internal stop codons, ensuring successful expression in the host system (Figure 7).

To evaluate potential biosafety concerns associated with ectopic expression of the ScWRKY5 construct, an in silico off-target screening was performed using multiple publicly available tools. The CRISPOR tool (http://crispor.tefor.net/) was employed to assess the ScWRKY5 CDS for potential CRISPR/Cas9 off-target sites within the Solanum lycopersicum genome. Additionally, siDirect v2.0 and miRanda were used to screen for potential siRNA and endogenous microRNA (miRNA) seed sequence complementarity, respectively.

The results revealed no significant off-target hits in annotated gene coding regions or regulatory elements of the tomato genome. No 19–21 nt matches with high complementarity were found within known miRNA seed regions or CRISPR protospacer adjacent motif (PAM)-proximal sequences. Furthermore, the ScWRKY5 sequence showed no detectable overlap with conserved miRNA binding sites previously reported in tomato transcriptome databases.

While off-target predictions using CRISPOR, siDirect, and miRanda detected no significant hits in coding regions or regulatory elements, whole-genome in silico T-DNA insertion site prediction was not performed. Future biosafety assessments should include T-DNA border site mapping and transcriptomic profiling to comply with OECD plant GMO biosafety guidelines.

Postoptimization, the ENc improved from 42.1 to 35.4, and RSCU analysis showed a significant shift toward preferred codons for Solanum lycopersicum, confirming successful bias reduction (Figure 8).

A homology-based structural model of the ScWRKY5 WRKY domain was generated using Swiss-Model/AlphaFold, revealing a compact globular fold with a four-stranded β-sheet engaging the WRKYGQK motif on the second strand and a C₂H₂-type zinc finger. Sequence alignment with WRKY domains from Arabidopsis, tomato, barley, and rice confirmed complete conservation of the motif and zinc-coordinating Cys/His residues, implying preservation of DNA-binding function via the characteristic β-wedge insertion into the W-box promoter sequence (Figure 10).

This study presents a comprehensive in silico strategy for codon optimization, cloning, and heterologous expression of the class III WRKY TF gene (ScWRKY5) from Saccharum officinarum into Solanum lycopersicum to enhance abiotic stress resilience. WRKY TFs, particularly class III members, are pivotal regulators in plant stress signaling pathways, modulating responses to drought, salinity, and heat stress through complex gene regulatory networks.^27,32,33

Codon optimization markedly improved the ScWRKY5 CDS’s CAI from 0.72 to 0.94 and GC content from 42.56% to 52.37%, indicating enhanced translational compatibility with S. lycopersicum. ENc and RSCU analyses confirmed a shift toward host-preferred codons, consistent with outcomes from prior optimization studies targeting stress-responsive genes.^{24–26,29–31,34,35}

The optimized gene was successfully in silico cloned into the pCAMBIA1302 vector under the control of the CaMV 35S promoter—a widely employed promoter for constitutive transgene expression in dicots.^28,36 The vector’s dual selection markers and mgfp5 reporter gene further enhance its suitability for heterologous expression studies. Simulated restriction digestion confirmed correct gene insertion, aligning with previous WRKY gene transfer studies.^37–39 Although the CaMV 35S promoter was selected for constitutive expression, recent studies indicate that tissue-specific or stress-inducible promoters can mitigate pleiotropic effects associated with continuous transgene expression.^40,41 Future work should compare ScWRKY5 performance under both constitutive and inducible regulatory systems in tomato.

Protein modeling and subcellular localization analyses predicted ScWRKY5 as a nuclear TF, consistent with its anticipated regulatory role in stress response pathways.^42,43 The workflow diagrams presented here enhance methodological transparency and reproducibility, reflecting best practices in contemporary bioinformatics-driven molecular biology.^44,45

This study contributes uniquely by optimizing ScWRKY5, a gene not previously characterized for heterologous expression in tomato. The achieved CAI of 0.94 surpasses those reported for OsWRKY45 and SiWRKY29 constructs,^46,47 indicating superior translational efficiency. Additionally, ScWRKY5 features distinct stress-responsive cis-elements and sequence motifs enhancing its potential utility in multifactorial abiotic stress scenarios, as supported by transcriptomic evidence in sugarcane according to Lu et al.¹⁷

Transcriptome-wide RNA-seq profiling in transgenic lines is essential to detect off-target transcriptional perturbations and unintended metabolic consequences. Coupled phenotypic and biochemical analyses should monitor for potential trade-offs and pleiotropic effects.^48–50

Recent advances in CRISPR-Cas9, base-editing, and prime-editing offer alternatives to conventional overexpression systems by enabling precise regulation of TFs or their promoter regions.^51–53 Future work could adapt the ScWRKY5 construct for inducible or genome-edited expression platforms, reducing risks associated with constitutive 35S-driven transgene expression.

While this study offers a comprehensive in silico framework for codon optimization, virtual cloning, and predicted heterologous expression of the ScWRKY5 gene from Saccharum officinarum in Solanum lycopersicum, several limitations must be acknowledged.

Firstly, the research is entirely computational, and no experimental validation has yet been performed. Although improvements in CAI, GC content, and codon usage bias suggest potential for enhanced gene expression in tomato, these theoretical predictions require empirical confirmation through laboratory-based expression assays, stable transformation studies, and functional phenotypic evaluations under controlled abiotic stress conditions.

Secondly, while the predicted subcellular localization and presumed nuclear role of ScWRKY5 were inferred from bioinformatics analyses, other important functional aspects such as protein-protein interactions, transcriptional regulatory networks, and downstream gene targets remain unexplored. Addressing these knowledge gaps is essential to fully elucidate the mechanistic role of ScWRKY5 in stress tolerance pathways.

Thirdly, although a preliminary in silico off-target analysis was performed to predict potential plant miRNA interactions and CRISPR seed region homologies, this assessment was limited to sequence-based screening. Comprehensive biosafety evaluation will require transcriptome-wide analyses, such as RNA-seq profiling, following stable transformation to detect potential unintended effects, pleiotropic responses, or metabolic disruptions associated with constitutive overexpression of a TF.

Finally, the codon optimization strategy in this study was exclusively designed for Solanum lycopersicum, and the compatibility of the optimized ScWRKY5 sequence with other economically important dicot species remains to be investigated. Broadening this scope in future studies would enhance the construct’s applicability for wider crop improvement initiatives.

Despite these limitations, the study provides a practical and replicable computational workflow, laying a strong foundation for subsequent empirical validation and translational research on ScWRKY5-mediated abiotic stress tolerance.

To substantiate the in silico predictions presented in this study, a phased experimental validation strategy is essential. Initial work should involve transient expression assays in Nicotiana benthamiana to assess ScWRKY5 protein localization, promoter activity, and stress-responsive expression profiles. This should be followed by the stable transformation of Solanum lycopersicum lines, with subsequent screening for transgene integration, expression levels, and abiotic stress tolerance under controlled greenhouse conditions.

In parallel, RNA-seq-based transcriptome profiling of transgenic lines will be necessary to detect potential off-target effects, transcriptional perturbations, and gene network alterations arising from constitutive ScWRKY5 overexpression. Comprehensive phenotypic, physiological, and metabolic assessments should accompany these molecular analyses to identify any unintended trade-offs or growth penalties.

Additionally, given recent advances in plant gene modulation technologies, future work should consider adapting ScWRKY5 deployment to genome-editing platforms such as CRISPR-Cas9, base-editing, or inducible promoter systems. These tools offer more precise and context-dependent regulation of stress-responsive genes, potentially mitigating the risks associated with continuous 35S-driven overexpression.

Finally, expanding the codon optimization pipeline to other high-value dicot crops and evaluating the cross-species compatibility of the optimized ScWRKY5 sequence would enhance the gene’s utility in broader agricultural biotechnology applications, particularly in the context of climate-resilient crop development.