In-silico identification and characterization of biotic and abiotic stress-responsive transcription factors in okra (Abelmoschus esculentus L.)

Introduction

Okra (Abelmoschus esculentus (L.) Moench) is an important vegetable crop belonging to the family Malvaceae and is widely cultivated in tropical and subtropical regions of the world. It is known by different local names, including lady's finger, gumbo, and bhindi. Although its precise origin remains debated, Abyssinia has been suggested as the primary center of origin (KHAJURIA, 2023; Davis, 2022) [15,7] . The genus Abelmoschus comprises both cultivated and wild species, among which A. esculentus is the most extensively grown. Okra is an allopolyploid species showing considerable variation in chromosome number and ploidy levels, reflecting its complex evolutionary history (Nieuwenhuis et al., 2024; Yadav et al., 2025) [24,38] . Nutritionally, okra is valued for its high content of dietary fiber, proteins, vitamins (A, B, and C), and essential minerals such as calcium, iron, and phosphorus (Gemede et al., 2015; Bawa & Badrie, 2016; Uwiringiyimana et al., 2024) [12,4,34] . In addition to its culinary importance, okra has industrial and medicinal value, with mature fibers used in paper production and traditional remedies for genitourinary disorders and dysentery (Gaur et al., 2025; Elkhalifa et al., 2021) [11,9] . Globally, okra occupies a significant area under cultivation, with India being the largest producer, contributing over 70% of the world's total production (J. Singh & Nigam, 2023; Tikoo et al., 2025) [31,33] . Despite its economic importance, okra productivity remains limited by multiple biotic and abiotic stresses. Among biotic stresses, viral diseases such as yellow vein mosaic virus and enation leaf curl virus (ELCV) cause severe yield losses ranging from 50% to 90% (Mubeen et al., 2021; Mohapatra, 2023; Sastry et al., 2025) [23,21,27] . ELCV belongs to the genus Begomoviral of the family Geminiviridae and is transmitted by the whitefly (Bemisia tabaci) in a semi-persistent manner (Idrees et al., 2024; Fiallo-Olivé et al., 2020) [14,10] . The association of monopartite begomoviruses with betasatellites further enhances disease severity in okra (A. K. Singh et al., 2021; Varma & Singh, 2024) [30,35] . Abiotic stresses such as drought, high temperature, and poor soil conditions further exacerbate yield instability, collectively posing major challenges to sustainable okra production. Plant responses to biotic and abiotic stresses are primarily regulated at the transcriptional level through complex gene regulatory networks. [2,6,29] . TFmediated regulation represents the major control point of gene expression, enabling plants to respond dynamically to developmental cues and environmental stimuli (Swift et al., 2022; Alvarez et al., 2021) [32,1] . In plants, TFs constitute approximately 8% of the total gene pool, highlighting their functional importance. Several TF families, including AP2/ERF, WRKY, NAC, MYB, bZIP, ARF, and B3, play critical roles in regulating stress-responsive pathways and plant development (Liu et [, 39, 37] . These TFs function through sequence-specific DNA binding and protein-protein interactions and often cooperate with chromatin-modifying complexes to regulate transcription (Morgunova & Taipale, 2017; Velthuijs et al., 2021) [22,36] . Alterations in TF activity and regulatory specificity are recognized as major contributors to evolutionary adaptation and stress tolerance in plants. Advances in genome sequencing and bioinformatics have enabled large-scale identification and classification of TFs across plant species. Databases such as PlantTFDB provide comprehensive information on TF families, domain architectures, and functional annotations (Blanc-Mathieu et al., 2024) [5] . In addition, motif discovery tools such as the MEME Suite facilitate the identification of conserved protein motifs and DNA-binding domains, providing insights into TF function and regulatory mechanisms (Machanick & Bailey, 2011) [20] . Despite the availability of such resources, systematic information on transcription factors associated with biotic and abiotic stress tolerance in okra remains limited. Therefore, the present study aims to perform an in-silico identification and characterization of transcription factors in okra, with particular emphasis on their potential roles in stress tolerance. The findings are expected to provide a valuable genomic resource for functional studies and to support future breeding and biotechnological efforts aimed at developing stress-resilient okra cultivars.

Materials and Methods

Data Source

Unigene sequences derived from transcriptomic data of okra (Abelmoschus esculentus L.) were obtained from the World Vegetable Center (AVRDC). These sequences constituted the primary dataset for identifying transcription factors associated with biotic and abiotic stress responses.

Identification of Transcription Factors

Transcription factors (TFs) were identified using PlantTFDB version 5.0, an integrative plant transcription factor database that classifies TFs based on conserved DNA-binding domains and family-specific signatures. Unigene sequences were uploaded in FASTA format to the TF prediction module. Prediction was performed using domain annotation and BLAST searches against curated plant TF datasets, particularly from Arabidopsis thaliana.

For each predicted TF, information on TF family, best-hit A. thaliana homolog, BLAST E-value, and functional annotation was retrieved. TFs showing significant sequence similarity and low E-values were shortlisted. Based on their established roles in stress tolerance, transcription factors belonging to the WRKY, NAC, ERF, and bZIP families were selected for downstream analyses.

Protein Motif Discovery

Conserved protein motifs within the selected transcription factors were identified using the MEME Suite, employing the GLAM2 (Gapped Local Alignment of Motifs) algorithm. GLAM2 is particularly suited for identifying gapped motifs characteristic of transcription factor DNA-binding domains. Protein sequences corresponding to the selected TFs were used as input, and motif discovery was performed using default parameters with multiple independent replicates to ensure motif consistency. Motifs were ranked based on GLAM2 scores, with higher scores indicating stronger motif conservation. Information regarding motif length, alignment, positional distribution, and marginal scores was recorded for further analysis.

Motif Comparison and Validation

To validate the functional relevance of the identified motifs, comparative motif analysis was conducted using the TOMTOM tool within the MEME Suite. Predicted motifs were compared against curated motif databases to identify similarity with known transcription factor binding motifs. Motifs exhibiting significant similarity and alignment scores were considered biologically relevant.

Data Output and Visualization

Output files, including transcription factor prediction tables, motif alignments, and motif comparison results, were generated in HTML and PNG formats and used for result interpretation and figure preparation.

Results and Discussion

Identification and Classification of Stress-Responsive Transcription Factors

Transcription factors associated with biotic and abiotic stress responses in okra were identified using the PlantTFDB webbased platform. Unigene sequences derived from okra transcriptomic data were screened to predict transcription factors based on conserved DNA-binding domains and family classification. Based on prior evidence of stress-responsive TF families in plants, particularly in Arabidopsis thaliana, four major TF families WRKY, NAC, ERF, and bZIP were selected for detailed analysis (S. Li et al., 2024) [19] . A total of 17 transcription factors were identified as putatively involved in stress responses in okra (Table 1). Among these, 10 TFs were associated with biotic stress tolerance, including eight WRKY and two NAC family members. WRKY and NAC transcription factors are well documented as central regulators of plant defense signalling, particularly in pathogen recognition and activation of downstream defense genes (Saha et al., 2024; Dong et al., 2024) [25,8] . The predominance of WRKY TFs observed in this study supports their conserved and prominent role in regulating biotic stress responses. In contrast, seven transcription factors were identified as associated with abiotic stress tolerance, comprising five ERF and two bZIP family members. ERF and bZIP TFs are known to regulate plant responses to drought, salinity, temperature extremes, and hormonal signaling pathways, particularly through abscisic acid-dependent and independent mechanisms (Banerjee & Roychoudhury, 2017; Hu et al., 2022) [3,13 . The presence of AP2/ERF-related subfamilies, including DREB and RAV, further highlights the importance of these TFs in regulating dehydration, heat shock, cold, and salt stress responses. BLAST analysis against A. thaliana revealed that okra TF unigenes showed significant homology with wellcharacterized transcription factors, with strong E-values ranging from 1.00E-11 to 1.00E-110. Functional annotations of best hits indicated involvement in hormone signaling, stress-responsive transcriptional regulation, and developmental control, suggesting functional conservation across species. These results demonstrate that okra possesses a diverse and conserved repertoire of transcription factors potentially involved in both biotic and abiotic stress adaptation. To further elucidate the functional properties of the identified transcription factors, conserved protein motifs were analyzed using the MEME Suite, specifically the GLAM2 algorithm. The same 17 unigene sequences identified as transcription factors were used for motif discovery. GLAM2 is particularly suitable for detecting gapped motifs, which are characteristic of transcription factor DNA-binding domains and regulatory regions (Schultheis et al., 2024) [28] . A total of 17 gapped protein motifs were identified across the WRKY, NAC, ERF, and bZIP transcription factor families (Figure 1). These motifs exhibited conserved aligned columns interspersed with insertions and deletions, reflecting structural flexibility while maintaining functional specificity. Such gapped motifs are known to contribute to DNA-binding affinity and protein-protein interactions, which are critical for transcriptional regulation (Santos, 2025) [26] . GLAM2 assigned motif scores based on alignment strength, with higher scores indicating stronger motif conservation. Site-specific scores further reflected the degree of similarity between individual sequences and the consensus motif (Figure 2). Family-specific motif patterns were observed, with WRKY motifs showing high conservation consistent with their welldefined WRKY DNA-binding domain, while ERF and bZIP motifs displayed greater variability, likely reflecting their involvement in multiple stress signalling pathways.

Identification and Characterization of Conserved Protein Motifs

Comparative Motif Analysis and Functional Implications

To validate the biological relevance of the identified motifs, motif comparison analysis was performed using the TOMTOM tool. TOMTOM analysis revealed that the predicted motifs showed strong similarity to known transcription factor binding motifs corresponding to WRKY, NAC, ERF, and bZIP families (Figure 3). This confirms that the identified motifs represent authentic regulatory elements involved in transcriptional control.

The conservation of these motifs across TF families suggests that okra transcription factors share regulatory mechanisms similar to those characterized in model plants. Given that transcription factors regulate stress responses by coordinating large gene networks, the presence of conserved motifs supports their potential role in orchestrating defense and stress-adaptive pathways.

Implications for Okra Improvement

The identification of stress-responsive transcription factors and their conserved motifs provides valuable insights into the transcriptional regulation of biotic and abiotic stress tolerance in okra. WRKY and NAC TFs identified in this study may serve as key candidates for improving disease resistance, particularly against viral pathogens. Similarly, ERF and bZIP TFs may be targeted for enhancing tolerance to drought, salinity, and temperature stress. These findings demonstrate the potential application of transcription factor genes in okra improvement through markerassisted selection, transgenic approaches, or genome editing strategies. Overall, this in-silico study establishes a foundational framework for future functional validation and utilization of transcription factors to develop stress-resilient okra cultivars.

Conclusion

A total of 962 transcription factors were identified from 153,414 okra unigenes through in-silico analysis, of which 17 key transcription factors belonging to WRKY, NAC, ERF, and bZIP families were selected based on significant homology with Arabidopsis thaliana. WRKY and NAC families were mainly associated with biotic stress responses, whereas ERF and bZIP families were linked to abiotic stress tolerance. Future functional validation of these transcription factors through expression analysis and genome editing may enable their effective deployment in developing stress-resilient okra cultivars.

Disclaimer (Artificial intelligence)

The author (s) hereby declare that NO generative AI technologies such as Large Language Models (ChatGPT, COPILOT, etc.) and text-to-image generators have been used during the writing or editing of this manuscript.

Competing Interests

Acknowledgement

The authors have declared that no competing interests exist. AcknowledgementThe authors sincerely acknowledge the Department of Genetics and Plant Breeding, Chaudhary Charan Singh University, Meerut, for providing access to the Bioinformatics Laboratory and necessary facilities to carry out this research work. And also, sincerely thank the World Vegetable Center (AVRDC) for generously providing the transcriptomic data, including unigene sequences of okra (Abelmoschus esculentus L.), used in this study.

References

1. Alvarez JM, Brooks MD, Swift J, Coruzzi GM. Time-based systems biology approaches to capture and model dynamic gene regulatory networks. Annu Rev Plant Biol. 2021;72(1):105-131.
2. Baillo EH, Kimotho RN, Zhang Z, Xu P. Transcription factors associated with abiotic and biotic stress tolerance and their potential for crops improvement. Genes. 2019;10(10):771-771.
3. Banerjee A, Roychoudhury A. Abscisic-acid-dependent basic leucine zipper (bZIP) transcription factors in plant abiotic stress. Protoplasma. 2017;254(1):3-16.
4. Bawa SH, Badrie N. Nutrient profile, bioactive components, and functional properties of okra (Abelmoschus esculentus (L.) Moench). Fruits Vegetables Herbs. 2016;365-409.
5. Blanc-Mathieu R, Dumas R, Turchi L, Lucas J, Parcy F. Plant-TFClass: A structural classification for plant transcription factors. Trends Plant Sci. 2024;29(1):40-51.
6. Chacón-Cerdas R, Barboza-Barquero L, Albertazzi FJ, Rivera-Méndez W. Transcription factors controlling biotic stress response in potato plants. Physiol Mol Plant Pathol. 2020;112:101527-101527.
7. Davis TW. Grower's guide: A review for sustainable production of okra (Abelmoschus esculentus) in West Africa and other regions. Int J Res Appl Sci Eng Technol. 2022;10(10):128-138.
8. Dong B, Liu Y, Huang G, Song A, Chen S, Jiang J, et al. Plant NAC transcription factors in the battle against pathogens. BMC Plant Biol. 2024;24(1):958-958.
9. Elkhalifa AEO, Alshammari E, Adnan M, Alcantara JC, Awadelkareem AM, Eltoum NE, et al. Okra (Abelmoschus esculentus) as a potential dietary medicine with nutraceutical importance for sustainable health applications. Molecules. 2021;26(3):696-696.
10. Fiallo-Olivé E, Pan LL, Liu SS, Navas-Castillo J. Transmission of begomoviruses and other whitefly-borne viruses: Dependence on the vector species. Phytopathology. 2020;110(1):10-17.
11. Gaur SS, Raveendran A, Athulya A, Narayanan SA, Shams R, Dash KK, et al. Exploring the nutritional, pharmacological, and industrial potential of okra and its byproducts: A comprehensive review. Discover Food. 2025;5(1):251-251.
12. Gemede HF, Ratta N, Haki GD, Woldegiorgis AZ, Beyene F. Nutritional quality and health benefits of okra (Abelmoschus esculentus): A review. J Food Process Technol. 2015;6:458-458.
13. Hu Y, Chen X, Shen X. Regulatory network established by transcription factors transmits drought stress signals in plant. Stress Biol. 2022;2(1):26-26.
14. Idrees MA, Abbas A, Saddam B, Bashir MH, Naveed H, Khan AK, et al. Persistence and circulative transmission of begomovirus by whitefly vectors: A comprehensive review. Int J Trop Insect Sci. 2024;44(2):405-417.
15. Khajuria A. Molecular and biochemical characterization of promising okra (Abelmoschus esculentus) genotypes. India; 2023.
16. Li S, Khoso MA, Xu H, Zhang C, Liu Z, Wagan S, et al. WRKY transcription factors as key regulators of plant resilience to environmental stresses. Agronomy. 2024;14(10):2421-2421.
17. Li T, Li B, Wang Y, Xu J, Li W, Chen Z, et al. WRKY transcription factors in rice: Key regulators orchestrating development and stress resilience. Plant Cell Environ. 2025;48(11):8388-8406.
18. Liu X, Zhou G, Chen S, Jia Z, Zhang S, Ren M, et al. Genome-wide analysis of the AP2/ERF gene family in Tritipyrum and salt-tolerance response of TtERF_B2-50. BMC Genomics. 2023;24(1):541-541.
19. Ma Z, Hu L, Jiang W. Understanding AP2/ERF transcription factor responses and tolerance to abiotic stresses in plants. Int J Mol Sci. 2024;25(2):893-893.
20. Machanick P, Bailey TL. MEME-ChIP: Motif analysis of large DNA datasets. Bioinformatics. 2011;27(12):16961697.
21. Mohapatra S. Evaluation of pest management modules for insect pest complex in okra (Abelmoschus esculentus (L.) Moench). Bhubaneswar: OUAT; 2023.
22. Morgunova E, Taipale J. Structural perspective of cooperative transcription factor binding. Curr Opin Struct Biol. 2017;47:1-8.
23. Mubeen M, Iftikhar Y, Abbas A, Abbas M, Zafar-ul-Hye M, Sajid A, et al. Yellow vein mosaic disease in okra (Abelmoschus esculentus L.): An overview. Phyton. 2021;90(6):1573-1587.
24. Nieuwenhuis R, Hesselink T, Van den Broeck HC, Cordewener J, Schijlen E, Bakker L, et al. Genome architecture and genetic diversity of allopolyploid okra (Abelmoschus esculentus). Plant J. 2024;118(1):225-241.
25. Saha B, Nayak J, Srivastava R, Samal S, Kumar D, Chanwala J, et al. Involvement of WRKY transcription factors in plant disease defense signaling. Planta. 2024;259(1):7-7.
26. Santos JRL. Unfolding structure from sequence: A computational perspective on genomic and molecular organization. Canada; 2025.
27. Sastry KS, Venkataravanappa V, Mahesha B, Reddy MK, Mahatma L. Virus diseases of okra. In: Tikoo SK, Angadi S, Tiwari A, Yadav RK, Tomar BS, Adeniyi AH, editors. Okra: Status, Challenges and Opportunities. Singapore: Springer Nature; 2025. p. 41-63.
28. Schultheis H, Bentsen M, Heger V, Looso M. Uncovering uncharacterized binding of transcription factors from ATAC-seq footprinting data. Sci Rep. 2024;14(1):92759275.
29. Sharma S, Kapoor S, Ansari A, Tyagi AK. General transcription factors of RNA polymerase II and their roles in plant development and stress Crit Rev Biochem Mol Biol. 2024;59(5):267-309.
30. Singh AK, Yadav BK, Krishna R, Kumar RV, Mishra GP, Karkute SG, et al. Bhendi yellow vein mosaic virus and betasatellite cause enation leaf curl disease in okra. Plant Dis. 2021;105(9):2595-2600.
31. Singh J, Nigam R. Importance of okra (Abelmoschus esculentus L.) and its proportion in the world as a nutritional vegetable. Int J Environ Clim Change. 2023;13(10):1694-1699.
32. Swift J, Greenham K, Ecker JR, Coruzzi GM, McClung CR. The biology of time: Dynamic responses of cell types to developmental, circadian and environmental cues. Plant J. 2022;109(4):764-778.
33. Tikoo SK, Angadi S, Tiwari A, Yadav RK, Tomar BS, Adeniyi AH, editors. Okra: Status, challenges and opportunities. Singapore: Springer Nature; 2025.
34. Uwiringiyimana T, Habimana S, Umuhozariho MG, Bigirimana VP, Uwamahoro F, Ndereyimana A, et al. Review on okra (Abelmoschus esculentus (L.) Moench) production, nutrition and health benefits. Rwanda J Agric Sci. 2024;3(1):71-87.
35. Varma A, Singh MK. Role of satellites in the evolution of begomoviruses. Viruses. 2024;16(6):970-970.
36. Velthuijs N, Meldal B, Geessinck Q, Porras P, Medvedeva Y, Zubritskiy A, et al. Integration of transcription coregulator complexes with DNA-binding factor interactomes. BBA Gene Regul Mech. 2021;1864(10):194749-194749.
37. Xiong H, He H, Chang Y, Miao B, Liu Z, Wang Q, et al. Multiple roles of NAC transcription factors in plant development and stress responses. J Integr Plant Biol. 2025;67(3):510-538.
38. Yadav RK, Vinay ND, Badiger M, Saakre M, Tomar BS, Choudhary H, et al. Okra breeding: History, milestones and challenges. In: Tikoo SK, Angadi S, Tiwari A, Yadav RK, Tomar BS, Adeniyi AH, editors. Okra: Status, Challenges and Opportunities. Singapore: Springer Nature; 2025. p. 95111.
39. Zheng C, Yang Q, Wang X, Chen Y, He R, Li X, et al. Transcription factors involved in plant stress and growth and development: NAC. Agronomy. 2025;15(4):949-949.