**4. Discussion**

In the mid-1980s, the relative water content (RWC) was introduced as the best reference point for the state of water in plants, expressing the balance between water absorption and consumption by transpiration [28]. The heat stress induces an increase of transpiration, with the e ffect of cooling and adapting the plant to heat. However a further transpiration dries up the tissues and the cells [29]. Generally, osmoregulation is one of the main mechanisms preserving turgor pressure in most plants against water loss; it causes the plant to continue to absorb water and maintain metabolic activity [30]. The RWC leaf may be the best biochemical growth/activity parameter that reveals the severity of stress [31]. Our data show a significant decrease in relative water content (RWC%) in both plants treated with di fferent temperatures, suggesting that both plants were under stress. *M. arvensis* L. var*. piperascens* has lost more water than *M.* × *piperita* L.

Heat stress disturbs the stable physiological condition in plants and for this reason scientists are trying to find a way to relieve stress. Brassinosteroids such as SA and M have recently been studied in relation to this issue. Coban and Baydar [32] have shown that brassinosteroids reduce salt stress. He et al. [33] inhibited heat stress in bluegrass using SA. In particular, SA stimulates the production and/or an increase of secondary metabolites from polyphenols by acting as an elicitor [34,35]. SA activates phenylalanine ammonia lyase (PAL) [36] and plays a role in the regulation of physiological processes [37]. M also alleviates stress damage, and this has been reported in cucumber in the germination phase [38] and in *Arabidopsis* in which it compensates for heat stress [39]. Our data show that both plants undergo a strong heat stress reducing RWC in a temperature-dependent way. Treatment with SA and M in both plants significantly reduce heat stress effect on RWC, confirming the protective role of these two hormones against heat stress.

In a previous work, we have shown that heat stress determines a change in oxygenated monoterpenes, monoterpene hydrocarbons, oxygenated sesquiterpenes, sesquiterpene hydrocarbons, and other components in *M.* × *piperita* L. (Mitcham variety) and *M. arvensis* L. (var. *piperascens*) essential oils [22]. In this study, brassinosteroids treatments in both the oils subjected to heat stress determined a variation in the composition of the essential oils and in the antioxidant enzymatic activity.

Saharkhiz and Goudarzi [40], showed that application of 150 mgL−<sup>1</sup> SA in *M.*×*piperita*L. significantly (*p* < 0.05) increased the oil content compared to control plants. In particular the treatment with different SA concentrations mostly increased menthone (15.8–18.1%) and menthol (46.3–47.4%) content.

In particular, monoterpenes (synthesized in Methylerythritol phosphate [MEP] pathway) and sesquiterpenes (synthesized in mevalonate [MVA] pathway) were the most important components. In the MEP pathway, menthol is synthesized in the cytoplasm and menthofuran is synthesized in the endoplasmic reticulum [41]. In particular, isopiperitenone from mitochondria is transferred to the cytoplasm and converts to pulegone. Pulegone can continue two branches of the MEP pathway: 1: It remains in the cytoplasm, is converted to menthone and finally to menthol; 2: it is transferred to the endoplasmic reticulum to be converted in menthofuran. So, in the MEP pathway, pulegone, menthone, menthofuran, and menthol have a crucial role and we should consider how they change under heat stress. In general, menthol and menthone have a significant negative correlation with menthofuran and pulegone, (menthol and menthone are reduced and the pulegone and menthofuran increased under the heat stress). Considering the (−)-Menthol biosynthesis pathway (Figure 6), we can hypothesize that under heat stress, pulegone reductase (PR) reduces its activity, leading to a decrease of the conversion of pulegone to menthone (that is the precursor of menthol). The increase of pulegone, due to the reduction of the activity of the enzyme that converts it in menthone, could also explain the increase of the mentofuran, which is synthesized from pulegone in the endoplasmic reticulum. In fact, pulegone had a significant positive correlation with menthofuran (Figure 6). In future studies, it will be necessary to verify the activity of the enzyme PR under heat stress and after treatment with the brassinosteroids.

As for the antioxidant activity, our studies have shown that SA and M have a positive effect on *M. arvensis* L. var*. piperascens* and *M.* × *piperita* L., increasing the activity of antioxidant enzymes in both species when used alone, but even more if applied simultaneously, demonstrating a synergistic effect.

On the other hand, an enhanced activity of CAT and SOD was observed in heat stressed plants of *Poa pratensis*, after the treatment with SA [33]. Xu et al. [42] reported that external M applications caused a significant increase in enzymatic antioxidants such as SOD, POX, CAT, and APX peroxidase and non enzymatic antioxidants such as ascorbic acid and vitamin E, resulting in decreased ROS levels and lipid peroxidation in cucumber under high temperature stress. Our data therefore not only confirms the effect of the two hormones on the activity of antioxidant enzymes and therefore the mitigating effect against the heat stress, but also show their ability to act in a synergistic way, which has not been demonstrated so far.

**Figure 6.** Menthol biosynthesis pathway. IPD: isopiperitenol dehydrogenase, IPR: isopiperitenone reductase, IPI: Isopentenyl diphosphate isomerase, PR: pulegone reductase, LH3:limonene 3-hydroxylase, MFS: menthofuran synthase, NMD: neomenthol reductase, MD: menthol dehydrogenases.
