**3. Results**

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

Significant decrease of Relative Water Contents (RWC%) in H2 and H3 suggested that plants could be under stress. *M. arvensis* L. var*. piperascens* lost more water than *M.* × *piperita* L. SA and M restored water content in a dose-dependant way (Figure 1). For this reason, for the next experiments, we only used the highest concentration for both treatments: melatonin 30 M (M3) and salicylic acid 4 mM (SA4).

**Figure 1.** Relative Water Content for (**a**) and (**c**) *M. arvensis* L. var*. piperascens* (Ma) and for (**b**) and (**d**) *M.* × *piperita* (Mp). Values are presented as means ± standard deviation (n = 15); values not accompanied by the same letter are significantly di fferent at *p* < 0.05, using the post-hoc Student–Newman–Keuls test. Lowercase letters(a–d) indicate significant di fferences between treatments for Ma; uppercase letters(A–C) indicate significant di fferences between treatments for Mp.

#### *3.2. Antioxidant Enzyme Activity*

As for the activity of antioxidant enzymes, heating determined a temperature-dependent increase, and treatments with SA4 and M3 determined a further increase, which proved to be extremely significant with the two hormones used simultaneously at their maximum concentrations.

In *M.* × *piperita*, the activity of all the measured antioxidant enzymes increased with increasing temperature both in the absence and presence of SA and M (M3, SA4). The only exception was the POX activity of samples C, which did not increase with increasing temperature but only under H3 conditions.

Moreover, in most cases the treatment with SA4 had a synergistic e ffect with the temperature compared to M3 on the activity of all the measured enzymes.

In general, for all the enzymatic activities measured, the samples treated with SA4M3 maintained a significantly higher enzyme activity compared to the C control samples and in the samples treated individually with SA and M3.

In *M. arvensis* L. var*. piperascens* the antioxidant enzymes activity in relation to temperature and treatment with M3, SA4, and M3SA4 followed the same trend shown in *M.* × *piperita* (Table 1).


**Table 1.** Enzyme activity in *M.* × *piperita* L. and *M. arvensis* L. var*. piperascens* for each treatment.

Values are presented as means ± standard deviation (n = 15); values not accompanied by the same letter(a–i,l,m), are significantly different at *p* < 0.05 using the post-hoc Student–Newman–Keuls test. For treatment details, see the Material and Methods Section 2.1.

#### *3.3. Essential Oil Yield*

Essential oil yields in *M.* × *piperita*and *M. arvensis* L. var*. piperascens* were not statistically different (Figure 2). Heat stress had a similar effect on both species, determining a significant reduction of essential oils as the temperature increased. In addition, both the treatments with SA4 and M3 determined an increase in the yield of essential oils in samples exposed to H3 heat stress conditions, even if there was no statistically significant difference between SA4 and M3.

**Figure 2.** Essential oil yield in *M. arvensis* L. var*. piperascens* (Ma) and *M.* × *piperita* (Mp) shown in H1, H2, and H3 conditions, and the effect of melatonin (M3) and salicylic acid (SA4) at their highest concentrations on essential oil yield in H3 condition.Values are presented as means ± standard deviation (n = 15); values not accompanied by the same letter are significantly different at *p* < 0.05, using the post-hoc Student–Newman–Keuls test. Lowercase letters(a–d) indicate significant differences between treatments for Ma; uppercase letters(A–D) indicate significant differences between treatments for Mp. For treatments details see Material and Methods Section 2.1).

The oxygenated monoterpenes amount in *M. arvensis* L. var*. piperascens*increased by using SA4, M3, and the two of them used simultaneously in normal condition (H1). In H2 conditions, only SA4 increased the oxygenated monoterpenes, while in H3 conditions only M3 increased them. In *M.* × *piperita* SA, M, and the two hormones used simultaneously increased the oxygenated monoterpenes in H1, and the major effect was observed by using M3. In H2 the oxygenated monoterpenes increased only by using S4 and M3 together. Unfortunately, we could not observe an increase of the oxygenated monoterpenes in H3 (Figure 3).

**Figure 3.** The amount of oxygenated monoterpenes in *M. arvensis* L. var*. piperascens* (Ma) and *M.* × *piperita* (Mp) under heat stress in H1, H2, and H3 and effects of melatonin (M3) and salicylic acid (SA4) on oxygenated monoterpenes. Values are presented as means ± standard deviation (n = 15); values not accompanied by the same letter are significantly different at *p* < 0.05, using the post-hoc Student-Newman-Keuls test. Lowercase letters(a–d) indicate significant differences between treatments for Ma; uppercase letters(A–G) indicate significant differences between treatments for Mp. For treatment details, see the Material and Methods Section 2.1.

A different trend was observed for monoterpene hydrocarbons on respect to oxygenated monoterpenes. In *M. arvensis* L. var*. piperascens* monoterpene hydrocarbons concentration was 4.3% for H1, 3.6% for H2, and 7.4% for H3 in control plants. Their amounts were decreased by using S4 (1.4%), M3 (1.3%), and both simultaneously (S4M3) (1.1%) in H1 conditions. In H2 they decreased by using M3 (0.8%), SA4 (0.8%), and the two of them simultaneously (1.0%), and the same applied for H3 by using SA4 (3.5%), M3 (2.5%) and the two of them simultaneously (4.8%). In *M.* × *piperita*the monoterpene hydrocarbons concentrations were in H1 4.0%, in H2 3.6% and in H3 5.2% in control plants. In H1 treatment by hormones decreased their amount (about 0.8%), and the same trend of reduction was observed for the other temperature conditions for all the treatments (data not shown).

Oxygenated sesquiterpenes were observed in very low concentrations in both the essential oils. Generally, in *M. arvensis* L. var*. piperascens* and in *M.* × *piperita* they were reduced or depleted by heat stress.

The dominant secondary metabolite in mint is menthol. Menthol dramatically decreased by heat stress, more than twice (in H1 56.6%, H2 39.0%, and H3 28.0%) in *M. arvensis* L. var*. piperascens* and about 4.5 fold (in H1 25%, H2 12.2%, and H3 5.6%) in *M.* × *piperita.* In *M. arvensis* L. var*. piperascens* only using SA4 and M3 simultaneously in H2 conditions the menthol concentration increased. In *M.* × *piperita* under H1 both SA4 and/or M3 increased the menthol concentration. In H3 by using SA4 and M3 simultaneously, the menthol concentration increased (14.3%) in comparison to control in H3 (Figure 4).

**Figure 4.** The amount of menthol in *M. arvensis* L. var*. piperascens* (Ma) and *M.* × *piperita* (Mp) under heat stress in H1, H2, and H3 and e ffect of melatonin (M3) and salicylic acid (SA4) on menthol. Values are presented as means ± standard deviation (n = 15); values not accompanied by the same letter are significantly different at *p* < 0.05, using the post-hoc Student-Newman-Keuls test. Lowercase letters(a–f) indicate significant di fferences between treatments for Ma; uppercase letters(A–F) indicate significant differences between treatments for Mp. For treatment details, see the Material and Methods Section 2.1.

Menthone is a precursor of menthol. The menthone concentration decreased in *M. arvensis* L. var. *piperascens* in H2 (7.6%) and H3 (12.0%), compared to H1 (14.5%). In *M.* × *piperita*the menthone concentration increased in H2 (15.9%) and decreased in H3 (9.5%), compared to H1 (13.0%). In normal condition (H1) in *M. arvensis* L. var. *piperascens* M3 decreased and SA4 increased menthone concentration. But in *M.* × *piperita*all the treatments increased menthone in H1 and H3 conditions. In H2 SA4 and M3 reduced menthone, but using both of them simultaneously increased menthone concentration (data not shown).

The menthofuran concentration in *M. arvensis* L. var*. piperascens* increased (in H1 7.8%, H2 34.0%, H3 13.0%) and in *M.* × *piperita*decreased (in H1 35.0%, H2 25.4%, H3 34.0%) under heat stress; the highest differences were observed in H2 condition. In *M. arvensis* L. var*. piperascens*, M3 increased, SA4 decreased, and the simultaneous use of both increased the menthofuran concentration, especially in the H1 condition. In H3 all treatments increased the menthofuran concentration. As regards *M.* × *piperita*, all treatments dramatically decreased the menthofuran concentration in H1. In H2, SA4 and M3 increased

the menthofuran concentration. In H3, all the treatments decreased the menthofuran concentration, especially during simultaneous use (data not shown).

The pulegone concentrations increased for both the essential oils under heat stress condition. Particularly, the pulegone concentrations in *M. arvensis* L. var*. piperascens* were in H1 5.6%, in H2 8.0%, and in H3 11.0% and for *M.* × *piperita* in H1 15.0%, in H2 24.3% and in H3 28.1%. In *M. arvensis* L. var*. piperascens* SA4 and the simultaneous use of the two brassinosteroids caused an increase in the pulegone concentration in normal condition (H1). In H2, all treatments increased the pulegone amount. In H3, M3 and the simultaneous use of multiple treatments increased pulegone. In *M.* × *piperita*, all treatments decreased the pulegone concentration in H1. But as for the heat conditions, in H2 all treatments increased the pulegone amount, while in H3 only M3 increased the pulegone concentration (Figure 5).

**Figure 5.** The amount of pulegone in *M. arvensis* L. var*. piperascens* (Ma) and *M.* × *piperita* (Mp) under heat stress in H1, H2, and H3 and effect of melatonin (M3) and salicylic acid (SA4) on pulegone. Values are presented as means ± standard deviation (n = 15); values not accompanied by the same letter are significantly different at *p* < 0.05, using the post-hoc Student-Newman-Keuls test. Lowercase letters(a–d) indicate significant differences between treatments for Ma; uppercase letters (A–G) indicate significant differences between treatments for Mp. For treatment details, see the Material and Methods Section 2.1.

In general, menthol has a significant negative correlation with menthofuran (r = −0.459 \*\*) and pulegone (r = –0.912 \*\*), that is to say that menthol is reduced and the pulegone increased under the heat stress. Also, menthofuran had a significant negative correlation with menthone (r = −0.527 \*\*). The pulegone had a significant positive correlation with menthofuran (r = 0.345 \*) (Table 2).

**Table 2.** Pearson correlation coefficients found among four important secondary metabolites (menthofuran, menthol, pulegone, and menthone) in the menthol pathway in *M. arvensis* L. var*. piperascens* and *M.* × *piperita*under the long-term extreme heat stress by using SA and M as a compensator of stress.


\*\* Correlation is significant at the 0.01 level (2-tailed).\* Correlation is significant at the 0.05 level (2-tailed).
