Exogenous Application of Hydrogen Peroxide, Hydrogen Sulfide, and Potassium Phosphite Enhances Rosmarinic Acid Accumulation and Modulates Key Biosynthetic Gene Expression in Melissa officinalis L.
[en] Melissa officinalis L. (lemon balm) is a well-known medicinal plant traditionally valued 16 for its pharmacological properties. Its aerial parts are rich in bioactive compounds such as 17 rosmarinic acid (RA), which play key roles in the plant defense system and contribute to 18 its antispasmodic, sedative, and memory-enhancing activities. However, the natural 19 concentrations of these secondary metabolites are typically low in medicinal plants. This 20 study investigated the influence of exogenous elicitors, hydrogen peroxide (HP, 5 and 10 21 mM), hydrogen sulfide (HS, 0.25 and 0.5 mM), and potassium phosphite (PP, 0.5 and 1 22 g/L) on the accumulation of photosynthetic pigments, antioxidant enzyme activity, 23 phenolic content, RA production, and the expression of RA biosynthesis-related genes. All 24 elicitors significantly increased chlorophyll a, b, and carotenoid levels. Treatment with HP 25 and HS notably elevated total phenolic and flavonoid contents. The activity of CAT and 26 APX enzymes varied depending on the elicitor type and concentration applied. Elicitation 27 enhanced RA accumulation by enhancing PAL activity and modulating the expression of 28 key biosynthetic genes (PAL, 4CL, TAT, and HPPR). HS treatment upregulated genes in 29 both phenylalanine and tyrosine pathways, whereas HP was more effective in stimulating 30 the tyrosine pathway. PP elicitation led to differential gene expression depending on its 31 concentration. These results demonstrate that HP, HS, and PP can modulate key metabolic 32 and signaling pathways, thereby boosting RA production and potentially enhancing the 33 medicinal value of M. officinalis in a safe and environmentally friendly manner.
Disciplines :
Chemistry Agriculture & agronomy
Author, co-author :
Abdi, Nahid
Ahmadi, Nima
Pakdin-Parizi, Ali
Fauconnier, Marie-Laure ; Université de Liège - ULiège > Département GxABT > Chemistry for Sustainable Food and Environmental Systems (CSFES)
Language :
English
Title :
Exogenous Application of Hydrogen Peroxide, Hydrogen Sulfide, and Potassium Phosphite Enhances Rosmarinic Acid Accumulation and Modulates Key Biosynthetic Gene Expression in Melissa officinalis L.
This work was supported by Ph.D. Student Grant Program by Tarbiat Modares University (TMU), Tehran, and Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari, Iran.
Ali, S., Tyagi, A., Bae, H., ROS interplay between plant growth and stress biology: challenges and future perspectives. Plant Physiol. Biochem., 203, 2023, 108032, 10.1016/j.plaphy.2023.108032.
Aroca, A., Gotor, C., Romero, L.C., Hydrogen sulfide signaling in plants: emerging roles of protein persulfidation. Front. Plant Sci., 9, 2018, 10.3389/fpls.2018.01369.
Barzotto, G.R., Cardoso, C.P., Jorge, L.G., Campos, F.G., Boaro, C.S.F., Foliar H2O2 application improve the photochemical and osmotic adjustment of tomato plants subjected to drought. Agriculture, 14, 2024, 1572, 10.3390/agriculture14091572.
Batista, P.F., Carlos, A., Silva, G.M., Almeida, M., Fernanda, E., Carolina, A., Rodrigues, A.A., Müller, C., Potassium phosphite induces tolerance to water deficit combined with high irradiance in soybean plants. Agronomy, 13, 2023, 382, 10.3390/agronomy13020382.
Belchí-Navarro, S., Rubio, M.A., Pedreño, M.A., Almagro, L., Production and localization of hydrogen peroxide and nitric oxide in grapevine cells elicited with cyclodextrins and methyl jasmonate. J. Plant Physiol. 237 (2019), 80–86, 10.1016/j.jplph.2019.03.013.
Blumenthal, M., Goldberg, A., Brinckmann, J., Herbal Medicine, Expanded Commission E Monographs. 2000.
Boeckx, T., Winters, A.L., Webb, K.J., Kingston-Smith, A.H., Polyphenol oxidase in leaves: is there any significance to the chloroplastic localization?. J. Exp. Bot. 66:12 (2015), 3571–3579, 10.1093/jxb/erv141.
Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (1976), 248–254, 10.1016/0003-2697(76)90527-3.
Carović-Stanko, K., Petek, M., Grdiša, M., Pintar, J., Bedeković, D., Ćustić, M.H., Satovic, Z., Medicinal plants of the family lamiaceae as functional foods-a review. Czech J. Food Sci. 34:5 (2016), 377–390, 10.17221/504/2015-CJFS.
Chang, C.C., Yang, M.H., Wen, H.M., Chern, J.C., Estimation of total flavonoid content in propolis by two complementary colometric methods. J. Food Drug Anal., 10, 2020, 10.38212/2224-6614.2748.
Corpas, F.J., Palma, J.M., H2S signaling in plants and applications in agriculture. J. Adv. Res. 24 (2020), 131–137, 10.1016/j.jare.2020.03.011.
Dai, J., Wen, D., Li, H., Yang, J., Rao, X., Yang, Y., Yang, J., Yang, C., Yu, J., Effect of hydrogen sulfide (H2S) on the growth and development of tobacco seedlings in absence of stress. BMC Plant Biol., 24, 2024, 162, 10.1186/s12870-024-04819-w.
Elufioye, T.O., Habtemariam, S., Hepatoprotective effects of rosmarinic acid: insight into its mechanisms of action. Biomed. Pharmacother., 112, 2019, 108600, 10.1016/j.biopha.2019.108600.
Fan, J., Hu, H., Lu, Y., Tao, L., Shen, X., Sun, C., Shen, Y., Mechanism by which exogenous H2O2 improves main secondary metabolites contents in post-harvest fresh ginkgo leaves through induced physiological responses mimicking stress. Ind. Crops Prod., 218, 2024, 118908, 10.1016/j.indcrop.2024.118908.
Fariduddin, Q., Khan, T.A., Yusuf, M., Hydrogen peroxide mediated tolerance to copper stress in the presence of 28-homobrassinolide in Vigna radiata. Acta Physiol. Plant. 36 (2014), 2767–2778, 10.1007/s11738-014-1647-0.
Ferriz-Martínez, R.A., Espinosa-Villarreal, N., Chávez-Servín, J.L., Mercado-Luna, A., Torre-Carbot, K., Serrano-Arellano, J., Saldaña, C., García-Gasca, T., Effect of foliar application of hydrogen peroxide macroconcentrations on growth parameters, phenolic compounds and antioxidant capacity in the leaves and seeds of Amaranthus hypochondriacus L. Plants, 12, 2023, 1499, 10.3390/plants12071499.
Fooladi Vanda, G., Shabani, L., Razavizadeh, R., Chitosan enhances rosmarinic acid production in shoot cultures of Melissa officinalis L. through the induction of methyl jasmonate. Bot. Stud., 60, 2019, 10.1186/s40529-019-0274-x.
Fotopoulos, V., Christou, A., Antoniou, C., Manganaris, G.A., Hydrogen sulphide: a versatile tool for the regulation of growth and defence responses in horticultural crops. J. Hortic. Sci. Biotechnol. 90 (2015), 227–234, 10.1080/14620316.2015.11513176.
Giani, M., Martínez-Espinosa, R.M., Carotenoids as a protection mechanism against oxidative stress in Haloferax mediterranei. Antioxidants, 9, 2020, 1060, 10.3390/antiox9111060.
Goldson, A., Lam, M., Scaman, C.H., Clemens, S., Kermode, A., Screening of phenylalanine ammonia lyase in plant tissues, and retention of activity during dehydration. J. Sci. Food Agric. 88 (2007), 619–625, 10.1002/jsfa.3126.
Gómez-Merino, F.C., Gómez-Trejo, L.F., Ruvalcaba-Ramírez, L., Trejo-Téllez, L.I., Application of phosphite as a biostimulant in agriculture. New and Future Developments in Microbial Biotechnology and Bioengineering, 2022, 135–153, 10.1016/B978-0-323-85581-5.00002-1.
Gondim, F.A., Gomes-Filho, E., Costa, J.H., Alencar, N.L.M., Prisco, J.T., Catalase plays a key role in salt stress acclimation induced by hydrogen peroxide pretreatment in maize. Plant Physiol. Biochem. 56 (2012), 62–71, 10.1016/j.plaphy.2012.04.012.
Gondim, F.A., Miranda, R.S., Filho, E.G., Prisco, J.T., Enhanced salt tolerance in maize plants induced by H2O2 leaf spraying is associated with improved gas exchange rather than with non-enzymatic antioxidant system. Theor. Exp. Plant Physiol. 25 (2013), 251–260, 10.1590/S2197-00252013000400003.
Hawrylak-Nowak, B., Dresler, S., Rubinowska, K., Matraszek-Gawron, R., Eliciting effect of foliar application of chitosan lactate on the phytochemical properties of Ocimum basilicum L. and Melissa officinalis L. Food Chem., 342, 2021, 128358, 10.1016/j.foodchem.2020.128358.
Hossain, M.A., Bhattacharjee, S., Armin, S.M., Qian, P., Xin, W., Li, H.Y., Burritt, D.J., Fujita, M., Tran, L.S.P., Hydrogen peroxide priming modulates abiotic oxidative stress tolerance: insights from ROS detoxification and scavenging. Front. Plant Sci., 6, 2015, 420, 10.3389/fpls.2015.00420.
İşeri, Ö.D., Körpe, D.A., Sahin, F.I., Haberal, M., Hydrogen peroxide pretreatment of roots enhanced oxidative stress response of tomato under cold stress. Acta Physiol. Plant. 35 (2013), 1905–1913, 10.1007/s11738-013-1228-7.
Jakovljević, D., Warchoł, M., Skrzypek, E., Rosmarinic acid as bioactive compound: molecular and physiological aspects of biosynthesis with future perspectives. Cells, 14(11), 2025, 850, 10.3390/cells14110850.
Jamaludin, R., Mat, N., Mohd, K.S., Badaluddin, N.A., Mahmud, K., Sajili, M.H., Khandaker, M.M., Influence of exogenous hydrogen peroxide on plant physiology, leaf anatomy and rubisco gene expression of the Ficus deltoidea jack Var. deltoidea. J. Agron., 10, 2020, 497, 10.3390/agronomy10040497.
Joshi, N.C., Yadav, D., Ratner, K., Kamara, I., Aviv-Sharon, E., Irihimovitch, V., Charuvi, D., Sodium hydrosulfide priming improves the response of photosynthesis to overnight frost and day high light in avocado (Persea americana mill, cv. 'hass'). Physiol. Plant. 168:2 (2020), 394–405, 10.1111/ppl.13023.
Kianersi, F., Amin Azarm, D., Pour-Aboughadareh, A., Poczai, P., Change in secondary metabolites and expression pattern of key rosmarinic acid related genes in Iranian lemon balm (Melissa officinalis L.) ecotypes using methyl jasmonate treatments. Molecules, 27(5), 2022, 1715, 10.3390/molecules27051715.
Kim, B.R., Jeong, Y.J., Kim, S., Kim, S.B., Lee, J., Lee, O.R., Kim, K.D., Jeong, J.C., Yang, B.W., Kim, C.Y., Elicitor-mediated enhancement of rosmarinic acid biosynthesis in cell suspension cultures of Lavandula angustifolia and in vitro biological activities of cell extracts. Plant Physiol. Biochem., 224, 2025, 109896, 10.1016/j.plaphy.2025.109896.
Koca, N., Karaman, S., The effects of plant growth regulators and L-phenylalanine on phenolic compounds of sweet basil. Food Chem. 166 (2015), 515–521, 10.1016/j.foodchem.2014.06.065.
Kwon, D.Y., Kim, Y.B., Kim, J.K., Park, S.U., Production of rosmarinic acid and correlated gene expression in hairy root cultures of green and purple basil (Ocimum basilicum L.). Prep. Biochem. 51:1 (2021), 35–43, 10.1080/10826068.2020.1789990.
Lichtenthaler, H.K., Buschmann, C., Extraction of phtosynthetic tissues: chlorophylls and carotenoids. Curr. Protoc. Food Anal. Chem. 1:F4.2 (2001), 1–F4.2.6, 10.1002/0471142913.faf0402s01.
Lisjak, M., Teklić, T., Wilson, I.D., Wood, M., Whiteman, M., Hancock, J.T., Hydrogen sulfide effects on stomatal apertures. Plant Signal. Behav. 6 (2011), 1444–1446, 10.4161/psb.6.10.17104.
Liu, F., Fu, X., Wu, G., Feng, Y., Li, F., Bi, H., Ai, X., Hydrogen peroxide is involved in hydrogen sulfide-induced carbon assimilation and photoprotection in cucumber seedlings. Environ. Exp. Bot., 175, 2020, 104052, 10.1016/j.envexpbot.2020.104052.
Livak, K.J., Schmittgen, T.D., Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25 (2001), 402–408, 10.1006/meth.2001.1262.
Ma, Q., Yang, J., Transcriptome profiling and identification of functional genes involved in H2S response in grapevine tissue cultured plantlets. Genes Genom 40 (2018), 1287–1300, 10.1007/s13258-018-0723-z.
Mayer, A.M., Harel, E., Ben-Shaul, R., Assay of catechol oxidase-a critical comparison of methods. Phytochemistry 5 (1966), 783–789, 10.1016/s0031-9422(00)83660-2.
Meda, A., Lamien, C.E., Romito, M., Millogo, J., Nacoulma, O.G., Determination of the total phenolic, flavonoid and proline contents in burkina fasan honey, as well as their radical scavenging activity. Food Chem. 91 (2005), 571–577, 10.1016/j.foodchem.2004.10.006.
Mejía-Teniente, L., Durán-Flores, F.D., Chapa-Oliver, A., Torres-Pacheco, I., Cruz-Hernández, A., González-Chavira, M., Ocampo-Velázquez, R., Guevara-González, R., Oxidative and molecular responses in Capsicum annuum L. after hydrogen peroxide, salicylic acid and chitosan foliar applications. Int. J. Mol. Sci. 14 (2013), 10178–10196, 10.3390/ijms140510178.
Mittler, R., Vanderauwera, S., Gollery, M., Van Breusegem, F., Reactive oxygen gene network of plants. Trends Plant Sci. 9 (2004), 490–498, 10.1016/j.tplants.2004.08.009.
Mofidnakhaei, M., Abdossi, V., Dehestani, A., Pirdashti, H., Babaeizad, V., Potassium phosphite affects growth, antioxidant enzymes activity and alleviates disease damage in cucumber plants inoculated with Pythium ultimum. Arch. Phytopathol. Pflanzenschutz 49 (2016), 207–221, 10.1080/03235408.2016.1180924.
Mohammadi, M.A., Han, X., Zhang, Z., Xi, Y., Boorboori, M.R., Wang-Pruski, G., Phosphite application alleviates pythophthora infestans by modulation of photosynthetic and physio-biochemical metabolites in potato leaves. Pathogens, 9, 2020, 170, 10.3390/pathogens9030170.
Mousavi, S.M., Shabani, L., Rosmarinic acid accumulation in Melissa officinalis shoot cultures is mediated by ABA. Biol. Plant. (Prague) 63 (2019), 418–424, 10.32615/bp.2019.057.
Nakano, Y., Asada, K., Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol. 28:1 (1987), 131–140, 10.1093/oxfordjournals.pcp.a077268.
Ngo, Y.L., Lau, C.H., Chua, L.S., Review on rosmarinic acid extraction, fractionation and its anti-diabetic potential. Food Chem. Toxicol. 121 (2018), 687–700, 10.1016/j.fct.2018.09.064.
Niu, L., Liao, W., Hydrogen peroxide signaling in plant development and abiotic responses: crosstalk with nitric oxide and calcium. Front. Plant Sci., 7, 2016, 230, 10.3389/fpls.2016.00230.
Nyanasaigran, L., Ramasamy, S., Gautam, A., Guleria, P., Kumar, V., Yaacob, J.S., Methyl jasmonate elicitation improves the growth performance and biosynthesis of antioxidant metabolites in Portulaca oleracea through ROS modulation. Ind. Crop. Prod., 216, 2024, 118709, 10.1016/j.indcrop.2024.118709.
Oksman-Caldentey, K.M., Inzé, D., Plant cell factories in the post-genomic era: new ways to produce designer secondary metabolites. Trends Plant Sci. 9:9 (2004), 433–440, 10.1016/j.tplants.2004.07.006.
Palma, J.M., Mateos, R.M., López‐Jaramillo, J., Rodríguez‐Ruiz, M., González‐Gordo, S., Lechuga‐Sancho, A.M., Corpas, F.J., Plant catalases as NO and H2S targets. Redox Biol., 34, 2020, 101525, 10.1016/j.redox.2020.101525.
Parveen, M., Asaeda, T., Rashid, M.H., Hydrogen sulfide induced growth, photosynthesis and biochemical responses in three submerged macrophytes. Flora 230 (2017), 1–11, 10.1016/j.flora.2017.03.005.
Paul, A., Acharya, K., Chakraborty, N., Involvement of phenylpropanoid pathway and shikimic acid pathway in environmental stress response. Biology and Biotechnology of Environmental Stress Tolerance in Plants, 2023, Apple Academic Press, 27–66.
Petrisor, G., Motelica, L., Craciun, L.N., Oprea, O.C., Ficai, D., Ficai, A., Melissa Officinalis: composition, pharmacological effects and derived release systems-A review. Int. J. Mol. Sci., 23(7), 2022, 3591, 10.3390/ijms23073591.
Qaiser, M.A., Khan, S., Qureshi, W.A., Haider, S.N.U.Z., Wang, W., Liu, Q., Eco-friendly sacrificial agents-free photocatalytic synthesis of hydrogen peroxide: recent innovations and future prospects. Int. J. Hydrogen Energy 95 (2024), 935–956, 10.1016/j.ijhydene.2024.11.272.
Ramezani, M., Rahmani, F., Dehestani, A., Comparison between the effects of potassium phosphite and chitosan on changes in the concentration of cucurbitacin E and on antibacterial property of Cucumis sativus. BMC Complement. Med. Ther., 17, 2017, 10.1186/s12906-017-1808-y.
Rastgoo, A., Chamani, E., Hir, Y.P., Mirjalili, M.H., Miran, M., Biochemical response and rosmarinic acid accumulation in callus culture of Salvia leriifolia benth (lamiaceae) irradiated with gamma rays. PCTOC, 161(3), 2025, 88, 10.1007/s11240-025-03110-6.
Salar, E., Khavari-Nejad, R.A., Mandoulakani, B.A., Najafi, F., Effects of TiO2 nanoparticles on activity of antioxidant enzymes, the expression of genes involved in rosmarinic acid biosynthesis and rosmarinic acid content in Dracocephalum kotschyi boiss. Russ. J. Plant Physiol. 68:1 (2021), 118–125, 10.1134/S1021443721010155.
Shakeri, A., Sahebkar, A., Javadi, B., Melissa officinalis L. -A review of its traditional uses, phytochemistry and pharmacology. J. Ethnopharmacol. 188 (2016), 204–228, 10.1016/j.jep.2016.05.010.
Trivellini, A., Lucchesini, M., Maggini, R., Mosadegh, H., Villamarin, T.S.S., Vernieri, P., Mensuali-Sodi, A., Pardossi, A., Lamiaceae phenols as multifaceted compounds: bioactivity, industrial prospects and role of "positive-stress. Ind. Crops Prod. 83 (2016), 241–254, 10.1016/j.indcrop.2015.12.039.
Trócsányi, E., György, Z., Zámboriné-Németh, É., New insights into rosmarinic acid biosynthesis based on molecular studies. Curr. Plant Biol., 23, 2020, 100162, 10.1016/j.cpb.2020.100162.
Ulgen, C., Yildirim, A.B., Sahin, G., Turker, A.U., Do magnetic field applications affect in vitro regeneration, growth, phenolic profiles, antioxidant potential and defense enzyme activities (SOD, CAT and PAL) in lemon balm (Melissa officinalis L.)?. Ind. Crops Prod., 169, 2021, 113624, 10.1016/j.indcrop.2021.113624.
Vafadar, F., Amooaghaie, R., Ehsanzadeh, P., Ghanadian, M., Talebi, M., Ghanati, F., Melatonin and calcium modulate the production of rosmarinic acid, luteolin, and apigenin in Dracocephalum kotschyi under salinity stress. Phytochemistry, 177, 2020, 112422, 10.1016/j.phytochem.2020.112422.
Valivand, M., Amooaghaie, R., Foliar spray with sodium hydrosulfide and calcium chloride advances dynamic of critical elements and efficiency of nitrogen metabolism in Cucurbita pepo L. under nickel stress. Sci. Hortic., 283, 2021, 110052, 10.1016/j.scienta.2021.110052.
Vinas, M., Mendez, J.C., Jiménez, V.M., Effect of foliar applications of phosphites on growth, nutritional status and defense responses in tomato plants. Sci. Hortic., 265, 2020, 109200, 10.1016/j.scienta.2020.109200.
Wang, Y., Li, L., Cui, W., Xu, S., Shen, W., Wang, R., Hydrogen sulfide enhances alfalfa (Medicago sativa) tolerance against salinity during seed germination by nitric oxide pathway. Plant Soil 351 (2011), 107–119, 10.1007/s11104-011-0936-2.
Woch, N., Laha, S., Gudipalli, P., Salicylic acid and jasmonic acid induced enhanced production of total phenolics, flavonoids, and antioxidant metabolism in callus cultures of Givotia moluccana (L.) sreem. In vitro Cell. Dev. Biol. Plant. 59:2 (2023), 227–248, 10.1007/s11627-023-10335-7.
Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., Madden, T.L., Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinf., 13, 2012, 10.1186/1471-2105-13-134.
Yıldız, M., Terzi, H., Bingül, N., Protective role of hydrogen peroxide pretreatment on defense systems and BnMP1 gene expression in Cr (VI)-stressed canola seedlings. Ecotoxicol. Environ. Saf. 22 (2013), 1303–1312, 10.1007/s10646-013-1117-2.
Zhang, H., Ye, Y.K., Wang, S.H., Luo, J.P., Tang, J., Ma, D.F., Hydrogen sulfide counteracts chlorophyll loss in sweet potato seedling leaves and alleviates oxidative damage against osmotic stress. J. Plant Growth Regul. 58 (2009), 243–250, 10.1007/s10725-009-9372-1.
Zhao, J., Davis, L.C., Verpoorte, R., Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol. Adv. 23 (2005), 283–333, 10.1016/j.biotechadv.2005.01.003.
Zou, H., Xiao, Q., Li, G., Wei, X., Tian, X., Zhu, L., Ma, F., Li, M., Revisiting the advancements in plant polyphenol oxidases research. Sci. Hortic., 341, 2025, 113960, 10.1016/j.scienta.2025.113960.
