De novo Transcriptomic Analysis in Salvia officinalis L. Exposed to Nitric Oxide

Abstract

Common sage (Salvia officinalis L.) is a medicinal and aromatic plant that belongs to the Lamiaceae family. Nitric oxide (NO) is a key signaling molecules in cells whose level are increased in response to various stimuli. Therefore, in order to identify the genes involved in NO signaling, de novo RNA sequencing (RNA-seq) strategy was employed to evaluate the transcriptional profiles in sage leaves. In this study, sage plants were foliar sprayed with 100 µM NO (donor diethylenetriamine NONOate). The leaf samples were then harvested after 24 hours of treatment for analyses. De novo assembly was performed on transcriptomic data using Trinity. Functional annotation and classification of the genes were performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. As a result, 69 genes were differentially expressed in NO-treated leaves as compared to control. Among them, 9 genes were up-regulated, while 60 genes were down-regulated. GO analysis of the differentially expressed genes (DEGs) indicated significant enrichment in categories such as response to stress, defense response, and response to external stimuli between the control and NO-treated seedlings. RNA-seq analysis provided basic data on which molecular mechanisms may be involved depending on the DEGs in sage exposed to NO.

Keywords

• Nitric oxide
• RNA-seq
• Salvia officinalis
• transcriptomics

References

1. 1. JT. Hancock, Nitric oxide signaling in plants, Plants, 9 (2020) 1550.
2. 2. M. Khan, S. Ali, T.N.I. Al Azzawi, S. Saqib, F. Ullah, A. Ayaz, W. Zaman, The key roles of ROS and RNS as a signaling molecule in plant-microbe interactions, Antioxidants, 12 (2023) 268.
3. 3. C. Bruand, E. Meilhoc, Nitric oxide in plants: Pro- or anti-senescence, J. Exp. Bot., 70 (2019) 4419-4427.
4. 4. Y.A. Henry, B. Ducastel, A. Guissani, Basic chemistry of nitric oxide and related nitrogen oxides, nitric oxide research from chemistry to biology: EPR spectroscopy of nitrosylated compounds, Springer Science & Business Media, 2012.
5. 5. C. Stöhr, W.R. Ullrich, Generation and possible roles of NO in plant roots and their apoplastic space, J. Exp. Bot., 53 (2002) 2293-2303.
6. 6. N.K. Rolly, Q.M. Imran, M. Shahid, M. Imran, M. Khan, S.U. Lee, A. Hussain, I.J. Lee, B.W. Yun, Drought-induced AtbZIP62 transcription factor regulates drought stress response in Arabidopsis, Plant Physiol. Biochem., 156 (2020) 384-395.
7. 7. K.J. Gupta, A.R. Fernie, W.M. Kaiser, J.T. van Dongen, On the origins of nitric oxide, Trends Plant Sci., 16 (2011) 160−168.
8. 8. Y.A. Leshem, Nitric oxide in plants: occurrence, function and use, Springer Science & Business Media, 2012.

9. 9. F.J. Corpas, S. González−Gordo, J.M. Palma, Nitric oxide: a radical molecule with potential biotechnological applications in fruit ripening, J. Biotech., 324 (2020) 211-219.
10. 10. M. Jakovljević, S. Jokic, M. Molnar, Bioactive profile of various Salvia officinalis L., Plants, 8 (2019) 55.
11. 11. T. Atsuko, O. Hiroshi, Phylogenetic relationships among subgenera, species, and varieties of Japanese Salvia L. (Lamiaceae), J. Plant Res., 124 (2011) 245-252.
12. 12. A. Ageeva-Kieferle, E. Georgii, B. Winkler, A. Ghirardo, A. Albert, P. Hüther, A. Mengel, C. Becker, J.P. Schnitzler, J. Durner, C. Lindermayr, Nitric oxide coordinates growth, development, and stress response via histone modification and gene expression, Plant Physiol., 187 (2021) 336-360.
13. 13. H. Terzi, H. Yalçın, M. Yıldız, G. Zengin, E. Pehlivan, A.I. Uba, Exogenous nitric oxide induces production of phenolic compounds enzyme inhibitory properties and antioxidant capacity through activating the phenylpropanoid pathway in sage (Salvia officinalis) leaves, S. Afr. J. Bot., 180 (2025) 811–819.
14. 14. S. Andrews, FastQC: A quality control tool for high throughput sequence data, (2010) https://github.com/s-andrews/FastQC
15. 15. A.M. Bolger, M. Lohse, B. Usadel, Trimmomatic: A flexible trimmer for Illumina Sequence data, Bioinformatics, 30 (2014) 2114-2120.
16. 16. M.G. Grabherr, B.J. Haas, M. Yassour, J.Z. Levin, D.A. Thompson, Full-length transcriptome assembly from RNA-Seq data without a reference genome, Nat. Biotechnol., 29 (2011) 644-652.
17. 17. R. Smith-Unna, C. Boursnell, R. Patro, J.M. Hibberd, S. Kelly, TransRate: reference-free quality assessment of de novo transcriptome assemblies, Genome Res., 26 (2016) 1134-1144.
18. 18. M. Manni, M.R. Berkeley, M. Seppey, E.M. Zdobnov, BUSCO: assessing genomic data quality and beyond, Curr. Protoc., 1 (2021) e323.
19. 19. B. Langmead, S.L. Salzberg, Fast gapped-read alignment with Bowtie2, Nat. Methods, 9 (2012) 357-359.
20. 20. B. Haas and A. Papanicolaou, TransDecoder 5.7.1 (2023), https://github.com/TransDecoder/TransDecoder/blob/master/TransDecoder.Predict
21. 21. N.L. Bray, H. Pimentel, P. Melsted, L. Pachter, Near-optimal probabilistic RNA-seq quantification, Nat. Biotechnol., 34 (2016) 525-527.
22. 22. M.I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Genome Biol., 15 (2014) 1-21.
23. 23. L. Kolberg, U. Raudvere, I. Kuzmin, J. Vilo, H. Peterson, Gprofiler2-an R package for gene list functional enrichment analysis and namespace conversion toolset g:Profiler, F1000Research, 9 (2020) 709.
24. 24. M. Ali, P. Li, G. She, D. Chen, X. Wan, J. Zhao, Transcriptome and metabolite analyses reveal the complex metabolic genes involved in volatile terpenoid biosynthesis in garden sage (Salvia officinalis), Sci. Rep., 7 (2017) 1-21.
25. 25. X. Lv, Y. Jin, Y. Wang, De novo transcriptome assembly and identification of salt-responsive genes in sugar beet M14, Comput. Biol. Chem.,75 (2018) 1-10.
26. 26. C. Liang, X. Liu, S.M. Yiu, B.L. Lim, De novo assembly and characterization of Camelina sativa transcriptome by paired-end sequencing, BMC Genomics, 14 (2013) 146. 27. P. Gahlan, H.R. Singh, R. Shankar, De novo sequencing and characterization of Picrorhiza kurrooa transcriptome at two temperatures showed major transcriptome adjustments, BMC Genomics, 13 (2012) 126.
27. 28. F. Xie, C.E. Burklew, Y. Yang, M. Liu, P. Xiao, B. Zhang, D. Qiu, De novo sequencing and a comprehensive analysis of purple sweet potato (Impomoea batatas L.) transcriptome, Planta, 236 (2012) 101-113.
28. 29. L. Yang, G. Ding, H. Lin, H. Cheng, Y. Kong, Y. Wei, X. Fang, R. Liu, L. Wang, X. Chen, C. Yang, Transcriptome analysis of medicinal plant Salvia miltiorrhiza and identification of genes related to tanshinone biosynthesis, PLoS One, 8 (2013) e80464.
29. 30. A. Riemenschneider, R. Wegele, A. Schmidt, J. Papenbrock, Isolation and characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana, FEBS J., 272 (2005) 1291-1304.
30. 31. Y. Zhang, F. Yun, X. Man, D. Huang, W. Liao, Effects of hydrogen sulfide on sugar, organic acid, carotenoid, and polyphenol level in tomato fruit, Plants, 12 (2023) 719.
31. 32. M. Ng, M.F. Yanofsky, Activation of the Arabidopsis B class homeotic genes by APETALA1, Plant Cell, 13 (2001) 739-753.
32. 33. C. Liu, J. Zhou, K. Bracha-Drori, S. Yalovsky, T. Ito, H. Yu, Specification of Arabidopsis floral meristem identity by repression of flowering time genes, Development, 134 (2007) 1901-1910.
33. 34. D. Weigel, E.M. Meyerowitz, Activation of floral homeotic genes in Arabidopsis, Science, 261 (1993) 1723-1726.
34. 35. G.B.G. Moorhead, L. Trinkle-Mulcahy, A. Ulke-Lemée, Emerging roles of nuclear protein phosphatases, Nat. Rev. Mol. Cell Biol.,8 (2007) 234–244.
35. 36. A. Schweighofer, I. Meskiene, Protein phosphatases in plant growth signalling pathways, in: L. Bögre, G. Beemster (Eds.), Plant Growth Signaling, Plant Cell Monographs, vol. 10, Springer, Berlin, 2008.
36. 37. A. Singh, S.K. Jha, J. Bagri, G.K. Pandey, ABA inducible rice protein phosphatase 2C confers ABA insensitivity and abiotic stress tolerance in Arabidopsis, PLoS One, 10 (2015) e0125168.
37. 38. C. Jung, H.N. Nguyen, C. Jong-Joo, Transcriptional regulation of protein phosphatase 2C genes to modulate abscisic acid signaling, Int. J. Mol. Sci., 21 (2020) 9517. 39. A. Schaller, A. Stintzi, S. Rivas, I. Serrano, N.V. Chichkova, A.B. Vartapetian, D. Martínez, J.J. Guiamét, D.J. Sueldo, R.A.L. van der Hoorn, V. Ramírez, P. Vera, From structure to function-a family portrait of plant subtilases, New Phytol., 218 (2018) 901-915.
38. 40. M. Budič, J. Sabotič, V. Meglič, J. Kos, M. Kidrič, Characterization of two novel subtilases from common bean (Phaseolus vulgaris L.) and their responses to drought, Plant Physiol Biochem., 62 (2013) 79-87.
39. 41. X. Duan, Z. Zhang, J. Wang, K. Zuo, Characterization of a novel cotton subtilase gene GbSBT1 in response to extracellular stimulations and its role in Verticillium resistance, PLoS One, 11 (2016) e0153988.
40. 42. R.A. Salzman, J.A. Brady, S.A. Finlayson, C.D. Buchanan, E.J. Summer, F. Sun, P.E. Klein, R.R. Klein, L.H. Pratt, M.M. Cordonnier-Pratt, J.E. Mullet, Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid, and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses, Plant Physiol., 138 (2005) 352-368.
41. 43. W.F. Chen, X.B.Wei, S. Rety, L.Y. Huang, N.N. Liu, S.X. Dou, X.G. Xi, Structural analysis reveals a “molecular calipers” mechanism for a LATERAL ORGAN BOUNDARIES DOMAIN transcription factor protein from wheat, J. Biol. Chem., 294 (2019) 142-156.
42. 44. A. Mangeon, E.M. Bell, W.C. Lin, B. Jablonska, P.S. Springer, Misregulation of the LOB domain gene DDA1 suggests possible functions in auxin signalling and photomorphogenesis, J. Exp. Bot., 62 (2011) 221-233.
43. 45. P. Yu, C. Gutjahr, C. Li, F. Hochholdinger, Genetic control of lateral root formation in cereals, Trends Plant Sci., 21 (2016) 951-961.
44. 46. Y. Jing, R. Lin, The VQ motif-containing protein family of plant-specific transcriptional regulators, Plant Physiol., 169 (2015) 371-378.
45. 47. Z. Lai, Y. Li, F. Wang, Y. Cheng, B. Fan, J.Q. Yu, Z. Chen, Arabidopsis sigma factor binding proteins are activators of the WRKY33 transcription factor in plant defense, Plant Cell, 23 (2011) 3824-3841.
46. 48. G. Castrillo, E. Sánchez-Bermejo, L. de Lorenzo, P. Crevillén, A. Fraile-Escanciano, T.C. Mohan, A. Mouriz, P. Catarecha, J. Sobrino-Plata, S. Olsson, Y.L. del Puerto, I. Mateos, E. Rojo, L.E. Hernández, J.A. Jarillo, M. Piñeiro, J. Paz-Ares, A. Leyva, WRKY6 Transcription factor restricts arsenate uptake and transposon activation in Arabidopsis, Plant Cell, 25 (2013) 2944-2957.
47. 49. S. Torre, M. Tattini, C. Brunetti, S. Fineschi, A. Fini, F. Ferrini, F. Sebastiani, RNA-Seq analysis of Quercus pubescens leaves: de novo transcriptome assembly, annotation and functional markers development, PLoS One, 9 (2014) e112487.
48. 50. R. Stracke, H. Ishihara, G. Huep, A. Barsch, F. Mehrtens, K. Niehaus, B. Weisshaar, Differential regulation of closely related R2R3-MYB transcription factors control flavonol accumulation in different parts of the Arabidopsis thaliana seedling, Plant J., 50 (2007) 660-677.
49. 51. B. Weisshaar, I.J. Gareth, Phenylpropanoid biosynthesis and its regulation, Curr. Opin. Plant Biol., 1 (1998) 251-257.