**1. Introduction**

The Lamiaceae family encompasses various genera, including aromatic herbs such as mint. Embracing half a dozen cultivated species, mint genus includes more than 30 species that are scattered worldwide, chiefly in temperate and tropical/subtropical regions. One of the distinctive features is that mint species possess essential oils [1].

Japanese mint or Cornmint (*Mentha arvensis* L. var. *piperascens* (Malinv. ex Holmes) Malinv. ex L.H.Bailey) is a fundamental natural source of monoterpenes, particularly L-menthol (up to 80% menthol), and it was already cultivated in ancient Japan as well as in China, India, and Brazil.

*Mentha* × *piperita* is an abortive hybrid of the species *M. aquatica* L. and *M. spicata.* Ecumenically, peppermint is one of the most commercial odorous scented herbs. The peppermint leaves have not only a peculiar, sweet, and strong odor, but also a redolent, warm, and spicy taste, with a cooling aftertaste. The supremacy of the essential oils of *Mentha* × *piperita* is due to the presence of menthone, isomenthone, and di fferent isomers of menthol. Nowadays, extensive usage of peppermint oil in flavoring chewing gums, sugar confectioneries, ice creams, desserts, baked goods, tobacco, and alcoholic beverages is just one of the most prevalent applications of such oils. Furthermore, it is also commonly employed in the flavoring of pharmaceutical and oral preparations [2].

Menthol shows various biological activities, such as sedative, anesthetic, antiseptic, gastric, and antipruritic. It is also one of the few natural monocyclic monoterpene alcohols that have characteristics conducive to fragrances. As such, it has been used to flavor various goods such as candies, chewing gums, and toothpaste [3,4].

Heat stress has effects on metabolite synthesis in aromatic plants, changing phenolic and antioxidants concentrations [5]. Heat stress induces the generation of reactive oxygen species (ROS), such as superoxide radicals (•O2 −), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH), in plants, thereby creating a state of oxidative stress in them. This increased ROS level in plants causes oxidative damage to biomolecules such as lipids, proteins, and nucleic acids, thus altering the redox homeostasis [6,7]. To avoid potential damage by ROS, a balance between production and elimination of ROS at the intracellular level must be regulated. This equilibrium between production and detoxification of ROS is sustained by enzymatic and nonenzymatic antioxidants [8,9]. The enzymatic components comprise several antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), guaiacol peroxidase (POX), and peroxiredoxins.

Salicylic acid (SA) and melatonin (M) are two brassinosteroids playing an important role in regulating physiological processes. SA is a phenolic compound with antioxidant properties, involved in the regulation of physiological processes in plants [10]. SA can modulate plant responses to a wide range of oxidative stresses [11]. When applied exogenously at suitable concentrations, SA was found to enhance the e fficiency of antioxidant system in plants [12]. M (N-acetyl-5-methoxytryptamine) is an indole hormone involved in multiple biological processes [13]. According to a lot of findings, M plays an important role in the regulation of plant growth and development [14] and provides a defense against abiotic stresses such as extreme temperature, excess copper, salinity, and drought [15–17]. A lot of studies have proven that M may act as a plant growth regulator in rooting, seed germination, and delay in leaf senescence and other morphogenetic features [18–20]. M has been observed to improve tolerance for multiple stresses including heat stress, and in particular, exogenous M treatments protect plants from temperature extremes [21].

The aim of this study was to evaluate the response of *M. arvensis* L. var*. piperascens* and *M.* × *piperita* to heat stress in relation to the production of essential oils in general and in particular of menthol, menthone, and isomenthone, which have considerable economic importance and play an important role in the industrial field. In particular, our goal was to investigate the potential of SA and M to mitigate the heat stress e ffects on the two plants, focusing on the variation of essential oils composition and antioxidant enzymes activity.

#### **2. Materials and Methods**

#### *2.1. Plant Material, Culture, and Treatment*

*M.* × *piperita* L. var. Mitcham and *M. arvensis* var. *piperascens* Malinv. ex L.H. Bailey were obtained from the "Safiabad agricultural and natural resources research and education center". Planting and cultivation conditions were carried out in the growth chambers. The method is described in detail in Heydari et al. [22].

After 40 culturing days, plants were sprayed with SA (2, 3, and 4 mM, reported as SA2, SA3, and SA4, respectively) and M (10 and 30 M, reported as M1 and M3), together with M and SA at the highest concentrations (M3SA4 ). Tap water was used for controls.

For each treatment, we selected 50 sample plants for subsequent experiments.

The abbreviation used to antioxidant enzyme activity and GC and GC-MS (Gas chromatography - Mass spectrometry) analysis are:

MpH1C = *M.* x *piperita* at the H1 temperature without treatment; MpH1M3 = *M.* x *piperita* at the H1 temperature treated with melatonin 3 mM; MpH1SA4 = *M.* x *piperita* at the H1 temperature treated with 4 mM salicylic acid; MpH1M3SA4 = *M.* x *piperita* at the H1 temperature treated with melatonin 3 mM and 4 mM salicylic acid; MpH2C = *M.* x *piperita* at the H2 temperature without treatment; MpH2M3 = *M.* x *piperita* at the H2 temperature treated with melatonin 3 mM; MpH2SA4 = *M.* x *piperita* at the H2 temperature treated with 4 mM salicylic acid; MpH2M3SA4 = *M.* x *piperita* at the H2 temperature treated with melatonin 3 mM and 4 mM salicylic acid; MpH3C = *M.* x *piperita* at the H3 temperature without treatment; MpH3M3 = *M.* x *piperita* at the H3 temperature treated with melatonin 3 mM; MpH3SA4 = *M.* x *piperita* at the H3 temperature treated with 4 mM salicylic acid; MpH3M3SA4 = *M.* x *piperita* at the H3 temperature treated with melatonin 3 mM and 4 mM salicylic acid; MaH1C = *M. arvensis* L. var*. piperascens* at the H1 temperature without treatment; MaH1M3 = *M. arvensis* L. var*. piperascens* at the H1 temperature treated with melatonin 3 mM; MaH1SA4 = *M. arvensis* L. var*. piperascens* at the H1 temperature treated with 4 mM salicylic acid; MaH1M3SA4= *M. arvensis* L. var*. piperascens* at the H1 temperature treated with melatonin 3 mM and 4 mM salicylic acid; MaH2C = *M. arvensis* L. var*. piperascens* at the H2 temperature without treatment; MaH2M3 = *M. arvensis* L. var*. piperascens* at the H2 temperature treated with melatonin 3 mM; MaH2SA4 = *M. arvensis* L. var*. piperascens* at the H2 temperature treated with 4 mM salicylic acid; MaH2M3SA4 = *M. arvensis* L. var*. piperascens* at the H2 temperature treated with melatonin 3 mM and 4 mM salicylic acid; MaH3C = *M. arvensis* L. var*. piperascens* at the H3 temperature without treatment; MaH3M3 = *M. arvensis* L. var*. piperascens* at the H3 temperature treated with melatonin 3 mM; MaH3SA4 = *M. arvensis* L. var*. piperascens* at the H3 temperature treated with 4 mM salicylic acid; MaH3M3SA4 = *M. arvensis* L. var*. piperascens* at the H3 temperature treated with melatonin 3 mM and 4 mM salicylic acid.

#### *2.2. Relative Water Content (RWC)*

*Relative Water Content* (RWC) was calculated according to Dhopte and Manuel [23]:

> RWC = (FW−DW)/(TW−DW) × 100

where FW is leaf fresh weight, DW is dry weight, and TW is leaf turgor mass of leaf samples [24] obtained measuring the leaf weight after 10–12 h in water saturating conditions.

#### *2.3. Antioxidant Enzyme Activity*

Protein extraction and the activity of antioxidant enzymes(SOD, CAT, GST ans PEROX) was carried out according to Maresca et al. [25].

#### *2.4. Isolation of Essential Oils*

Samples' shoots were air-dried in dark conditions at room temperature and were used for essential oils extraction. Each sample (50 g in three replications) was extracted using hydro-distillation for 3 h and Clevenger-type apparatus based on the standard procedure described by Russo et al. [26]. The essential oils were obtained with different yields (0.97 ± 0.02–3.26 ± 0.02%) on dry mass (w/w) and results were yellowish with a pleasant smell. The oils were dried with anhydrous sodium sulfate and stored under N2 at +4 ◦C in the dark for subsequent tests and analyses.

#### *2.5. GC and GC-MS Analysis*

Analytical gas chromatography was carried out on a Perkin-Elmer Sigma 115 gas chromatograph fitted with an Agilent HP-5 MS capillary column (30 m × 0.25 mm), 0.25 μm film thickness. The analysis was also performed by using a fused silica HP Innowax polyethylene glycol capillary column (50 m × 0.20 mm), 0.20 μm film thickness. Gas chromatography analysis was performed as done previously and described in detail by Rigano et al. [27]. Compounds identification and components relative percentages were carried out as described by Rigano et al. [27].
