Analysis of TYDC2 gene expression in the root, leaf, and stem tissues and its correlation with the rate of callus formation in the medicinal plant poppy (Papaver somniferum L.)

Subject Areas : Medicinal Plants

Nazila Bagheri ^{1 *} , Alireza Tari-nejad ² , mohammad majidi ³ , کریم حسن پور ⁴

1 - Faculty of Agriculture, Shahid Madani University of Tabriz
2 - Department of Biotechnology, Shahid Madani University of Azerbaijan, Tabriz, Iran
3 - عضو هیات علمی
4 - University of Tabriz

Received: 2024-09-04 Accepted : 2024-10-29 Published : 2024-12-06

Keywords: auxin hormone, callus formation, gene expression, sequencing ,

Abstract :

Poppy, scientifically known as *Papaver somniferum* L., is a member of the Papaveraceae family. This plant contains over 40 different alkaloids, with the most significant being morphine, codeine, thebaine, noscapine, and papaverine. The purpose of this research is to identify the expression level of the TYDC2 gene in the stem, root, and leaf tissues of the poppy medicinal plant using Next Generation Sequencing (NGS) and to evaluate its relationship with the rate of callus formation in these tissues. Investigating the expression of the TYDC2 gene can provide insights into the biological mechanisms involved in callus formation and the development of various plant tissues. Additionally, it may help improve techniques for tissue culture and the production of medicinal plants. In this study, the sequence of the TYDC2 gene in the three tissues (stem, root, and leaf) was selected from the Gene section of the NCBI website and downloaded using the SRA Toolkit. The quality of the data was assessed with FastQC, and a heatmap diagram was created using RStudio software, along with other gene expression comparison charts. Next, Murashige and Skoog culture medium was prepared with 2,4-D hormone at a concentration of 2 mg/liter. The mentioned tissues were placed in this medium, and the percentage of callus formation, along with the diameter and weight of the callus, was measured and compared with the level of gene expression. Finally, the gene was analyzed using gene ontology via g: profiler. The results indicated that the expression levels of the TYDC2 gene varied across the stem, root, and leaf tissues of the poppy. The highest expression level was found in the leaf tissue, while the lowest was observed in the root tissue. Additionally, the results of the callus formation with the 2,4-D hormone (2 mg/liter) in vitro showed that callus formation was highest in the leaf tissue and lowest in the root tissue, indicating a direct relationship between gene expression and the amount of callus formed in these tissues. This research contributes to the advancement of scientific knowledge in plant genetics and biotechnology and serves as a reference for future studies in this field. 

References:

Aghaali, Z., & Naghavi, M. R. (2024). Developing benzylisoquinoline alkaloid-enriched opium poppy via CRISPR-directed genome editing: A review. BMC Plant Biology, 24(1), 700.
Aghaali, Z., Naghavi, M. R., & Zargar, M. (2024). Collaboration of hairy root culture and scale-up strategies for enhancing the biosynthesis of medicinal and defensive alkaloids in Papaver sp. Current Plant Biology, 100381.
Alqethami, A., & Aldhebiani, A. Y. (2021). Medicinal plants used in Jeddah, Saudi Arabia: phytochemical screening. Saudi Journal of Biological Sciences, 28(1), 805-812.
Arumuganathan, K., & Earle, E. (1991). Nuclear DNA content of some important plant species. Plant molecular biology reporter, 9, 208-218.
Bagheri, N., Maleki Zanjani, B., & Ammarlou, A. (2020). A callus study of different explants of the medicinal Myrtle plant (Myrtus communis). Journal of Medicinal Plants Biotechnology, 6(1), 93-101.
Birnie, R., Bryce, S. D., Roome, C., Dussupt, V., Droop, A., Lang, S. H., . . . Stower, M. J. (2008). Gene expression profiling of human prostate cancer stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix interactions. Genome biology, 9, 1-13.
Cánovas, R., Moffat, A., & Turpin, A. (2016). CSAM: compressed SAM format. Bioinformatics, 32(24), 3709-3716.
Consortium, G. O. (2008). The gene ontology project in 2008. Nucleic acids research, 36(suppl_1), D440-D444.
D'Antuono, L. F., Moretti, A., & Lovato, A. F. (2002). Seed yield, yield components, oil content and essential oil content and composition of Nigella sativa L. and Nigella damascena L. Industrial crops and products, 15(1), 59-69.
Diaz-Bárcena, A., & Giraldo, P. (2023). Exploring the research evolution of Papaver somniferum and Cannabis sativa: A bibliometric comparative analysis. Industrial Crops and Products, 203, 117143.
Eckert, R. L., Crish, J. F., Efimova, T., Dashti, S. R., Deucher, A., Bone, F., . . . Balasubramanian, S. (2004). Regulation of involucrin gene expression. Journal of Investigative Dermatology, 123(1), 13-22.
Ghasemi, S., Kumleh, H. H., & Kordrostami, M. (2019). Changes in the expression of some genes involved in the biosynthesis of secondary metabolites in Cuminum cyminum L. under UV stress. Protoplasma, 256, 279-290.
Hori, K., Yamada, Y., Purwanto, R., Minakuchi, Y., Toyoda, A., Hirakawa, H., & Sato, F. (2018). Mining of the uncharacterized cytochrome P450 genes involved in alkaloid biosynthesis in California poppy using a draft genome sequence. Plant and Cell Physiology, 59(2), 222-233.
Kanei, M., Horiguchi, G., & Tsukaya, H. (2012). Stable establishment of cotyledon identity during embryogenesis in Arabidopsis by ANGUSTIFOLIA3 and HANABA TARANU. Development, 139(13), 2436-2446.
Lee, D.-K., Geisler, M., & Springer, P. S. (2009). LATERAL ORGAN FUSION1 and LATERAL ORGAN FUSION2 function in lateral organ separation and axillary meristem formation in Arabidopsis.
Lee, M.-L. T., Kuo, F. C., Whitmore, G., & Sklar, J. (2000). Importance of replication in microarray gene expression studies: statistical methods and evidence from repetitive cDNA hybridizations. Proceedings of the National Academy of Sciences, 97(18), 9834-9839.
Leite Dias, S., & D’Auria, J. C. (2024). The bitter truth: how insects cope with toxic plant alkaloids. Journal of Experimental Botany, erae312.
Májer, P., & Németh, É. Z. (2024). Alkaloid Accumulation and Distribution within the Capsules of Two Opium Poppy (Papaver somniferum L.) Varieties. Plants, 13(12), 1640.
Marioni, J. C., Mason, C. E., Mane, S. M., Stephens, M., & Gilad, Y. (2008). RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome research, 18(9), 1509-1517.
Miranda, M., Vega-Gálvez, A., Quispe-Fuentes, I., Rodríguez, M. J., Maureira, H., & Martínez, E. A. (2012). Nutritional aspects of six quinoa (Chenopodium quinoa Willd.) ecotypes from three geographical areas of Chile. Chilean journal of agricultural research, 72(2), 175.
Ngernsaengsaruay, C., Leksungnoen, N., Chanton, P., Andriyas, T., Thaweekun, P., Rueansri, S., . . . Hauyluek, W. (2023). Morphology, taxonomy, anatomy, and palynology of the opium poppy (Papaver somniferum L.) cultivation in Northern Thailand. Plants, 12(11), 2105.
Romero, I. G., Ruvinsky, I., & Gilad, Y. (2012). Comparative studies of gene expression and the evolution of gene regulation. Nature Reviews Genetics, 13(7), 505-516.
Sakai, H., Honma, T., Aoyama, T., Sato, S., Kato, T., Tabata, S., & Oka, A. (2001). ARR1, a transcription factor for genes immediately responsive to cytokinins. Science, 294(5546), 1519-1521.
Santelia, D., Vincenzetti, V., Azzarello, E., Bovet, L., Fukao, Y., Düchtig, P., . . . Geisler, M. (2005). MDR-like ABC transporter AtPGP4 is involved in auxin-mediated lateral root and root hair development. FEBS letters, 579(24), 5399-5406.
Shinozaki, K., Yamaguchi-Shinozaki, K., & Seki, M. (2003). Regulatory network of gene expression in the drought and cold stress responses. Current opinion in plant biology, 6(5), 410-417.
Tilkat, E., Ayaz Tilkat, E., Akkaya, Ö., & Özden Çiftçi, Y. (2024). Metabolic Engineering for Overproduction of Plant Secondary Metabolites: Alkaloids. Innovative Methods in Horticultural Crop Improvement: Molecular Tools, Nanotechnology and Artificial Intelligence, 297-328.
Vadhel, A., Bashir, S., Mir, A. H., Girdhar, M., Kumar, D., Kumar, A., . . . Mohan, A. (2023). Opium alkaloids, biosynthesis, pharmacology and association with cancer occurrence. Open Biology, 13(5), 220355.
Yoshikawa, T., & Furuya, T. (1983). Regeneration and in vitro flowering of plants derived from callus cultures of opium poppy (Papaver somniferum). Experientia, 39, 1031-1033.