Elucidation of the role of tetraspanin(TET) gene family in stress response of piper nigrum L.

Abstract

The study entitled “Elucidation of the role of Tetraspanin (TET) gene family in stress response of Piper nigrum L.” was conducted in the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram from 2023-2024. Tetraspanins are evolutionary conserved transmembrane proteins present in all multicellular organisms and they act as key modulators of signaling cascades and respond to cold, drought, and pathogen stress. Tetraspanins also play a crucial role in the biogenesis of extracellular vesicles (EVs) that facilitate the transport of various biomolecules such as secondary metabolites, small RNAs, short peptides, etc. They are the key players in the stress response of plants, as they interact with signaling molecules and target intracellular locations. The objective of the study was the characterization of the tetraspanin (TET) gene family in the stress response of Piper nigrum L. In this study, twelve tetraspanin gene candidates were identified from the P. nigrum via genome wide analysis. The P. nigrum tetraspanin genes identified belong to six distinct classes namely TET1, TET2, TET3, TET6, TET8, and TET10. The length of the genes ranged from 798 to 1491 bp, with encoded protein of 265- 496 amino acids. The deduced PnTET proteins were basic in nature with molecular weight ranging from 29.3 to 53.5 kDa. The characteristic motifs and residues were detected in all the identified P. nigrum tetraspanin proteins. Phylogenetic analysis segregated the Piper nigrum tetraspanins into six distinct paralogous clades. A detailed in silico analysis revealed cis-regulatory elements involved in stress response in the promoter region of PnTET genes. The expression profiling of selected PnTET genes such as PnTET8.1, PnTET3.1, and PnTET10 were carried out from the biotic and abiotic stressed P. nigrum variety Panniyur1 plants. The biotic stress was induced by the artificial inoculation of Phytophthora capsici in the leaves and roots of the plants and total RNA was isolated from the infected plants at 24hpi, 48hpi (early response), and 72hpi, 96hpi, 120hpi (late response). PnTET3.1 and PnTET8.1 were upregulated from 48 to 120 hours post-infection (Day 5) in the leaf and root of P. capsici-infected plants. The expression of PnTET10 showed a late upregulation in the root of the infected plant after 96hpi (1.71fold) meanwhile, an early upregulation at 24hpi (14.07 fold) was observed in the infected leaf. Although the upregulation of PnTET10 was observed in the subsequent stages in the infected leaf, the expression was found to be low compared to its early (24hpi) induction. Expression profiling of the selected three genes ie. PnTET3.1, PnTET8.1, and PnTET10 were also carried out from the leaves and roots of black pepper plants subjected to abiotic stress by 105 withholding irrigation for 10 days. PnTET3.1 showed the highest upregulation in leaves (2.53 fold) and roots (3.05 fold) of the abiotic stressed plant; while PnTET8.1 showed an upregulation in the leaves and roots with a fold change of 1.25 and 1.63 respectively. An upregulation of PnTET10 was observed in leaves and roots with a fold change of 1.47 and 1.32 respectively. The sequence characterization of Indian cultivated variety, Panniyur-1 PnTET3.1, PnTET8.1, and PnTET10 revealed the three-dimensional structure of their protein and typical magnoliid characteristics via phylogenetic analysis. The present study is the first report on the identification of tetraspanin (TET) gene family from black pepper. The genome-wide analysis found six distinct classes of tetraspanin genes in black pepper. The differential expression of PnTET3.1, PnTET8.1, and PnTET10 genes in stress conditions as well as the presence of stress-responsive regulatory components in the PnTET promoter regions indicated its functional role in black pepper. Future studies will be directed towards the elucidation of their role in defense responses in the susceptible and resistant varieties of black pepper.