*3.6. Leaf Minerals*

The effects of the factors considered (CV and S) and their interaction (CV × S) on the accumulation of minerals in basil leaves are shown in Table 5 and Figure 7. The concentrations of nitrate, Ca, Mg, Na, and Cl were influenced by the average CV and S effects and the CV × S interaction, except for K and P, where a significant difference was observed only for the average CV and S effects. Regardless of salt treatment, 'Lemon' had the highest P value (11.75 g kg−<sup>1</sup> dw) while 'Anise' had an 8.7% higher K concentration than 'Cinnamon' (Table 5). Regardless of the cultivar, compared to the control, salt stress significantly reduced nitrate, K, and P concentrations (Table 5), while an opposite trend was observed for Cl (Figure 7). Compared to the CV × S interaction, the salt treatment cultivars 'Cinnamon' and 'Lemon' had significantly higher Na concentrations than the Control (Figure 7). The same treatment increased Ca and Mg concentrations in 'Anise' and 'Cinnamon' (Table 5).

**Table 5.** Effect of basil cultivars (CV) and stress (S) on Nitrate, K, P, Ca, and Mg leaf concentration.


Data are mean values ± standard error, *n* = 3. Mean comparisons were performed by Tukey HSD test for CV and by *t*-Test for S. Different letters within each column indicate significant mean differences. ns, \*, \*\*, and \*\*\* denote non-significant or significant effects at *p* ≤ 0.05, 0.01 and 0.001, respectively.

**Figure 7.** Effect of Cultivar × Stress on Na (**A**) and Cl (**B**) leaf concentration. Data are mean values ± standard error, *n* = 3. Statistical significance of the CV × S interaction was determined by Tukey HSD test for Cultivar and by *t*-Test for Stress. Different letters indicate significant mean differences. \*\* and \*\*\* denote non-significant or significant effects at *p* ≤ 0.01 and 0.001, respectively.

#### **4. Discussion**

The significant difference recorded for total fresh weight in the basils evaluated in the present work underlines the strong impact of genotype. This variability was expected, assuming the phylogenetic gap between species and cultivars of the genus Ocimum. Regardless of salt treatment (Salt), 'Cinnamon' was the most productive cultivar, followed by 'Anise' and 'Lemon' (Figure 2). However, it should be noted that 'Anise' and 'Cinnamon' did not differ significantly in height, number of leaves, and leaf area, although the total fresh weight was different (Figure 3A–C). Relative to total fresh weight, the significant differences observed between cultivars did not result in a different response to salt stress, unlike what Ciriello et al. [27] observed in a recent work on basil. On average, salt treatment reduced the total fresh yield by 51.54% compared to the control; similar results were reported on *Salvia officinalis* L. [28] and *Mentha spicata* L. [29]. Regardless of cultivar, an adaptive response to salt stress was observed, culminating in reduced growth, an aspect that would allow plants to conserve energy by promoting the initiation of targeted defensive responses aimed at reducing permanent damage, as inferred from photosynthetic parameters shown in Table 2. Although Attia et al. [7] considered inappropriate to compare leaf production and photosynthetic activity (as it was related to a small number of leaves), in our study, salt stress reduced the net CO2 assimilation rate and transpiration. Like Bekhradi et al. [30] and Attia et al. [7], salt-induced osmotic and nutritional stress

would prompt plants to reduce stomatal conductance, affecting RuBisCo activity. Although salt stress can cause severe damage to photosystems due to poor management of excess excitation energy, the plants' morphophysiological and biochemical adaptations mitigated the deleterious effects induced by stress, as confirmed by Fv/Fm values that never reached critical values (Figure 4D). However, it is essential to note that, although Fv/Fm is the most widely used parameter for evaluating the performance of PSII under stress conditions [31], it may not be an acceptable parameter for assessing the physiological responses of plants under salt stress.

The analysis of morphological parameters of the salt-treated plants showed a lower sensitiveness of the Anise and Cinnamon cultivars than 'Lemon'. The latter reduced leaf area and height by 57.38 and 35.37%, compared to the control; in contrast, 'Anise' and 'Cinnamon' reduced, on average, the same parameters by 33.28 and 19.87%. The above is confirmed by the reduction in total dry weight in 'Lemon' (56.97%) in the salt treatment compared to the control. In contrast, no significant changes were observed in 'Cinnamon' for this key parameter. This result could be partially attributable to a constitutive higher tolerance to salt stress and partially to the increase in total dry matter, which increased from 9.27% (Cinnamon × Control) to 15.10% (Cinnamon × Salt) (Figure 3E). As Bernstein et al. [32] suggested, good hydration of plant tissues is a key characteristic of salt stress tolerance. However, in the present study, the unchanged values of total dry weight in 'Cinnamon' cannot be considered indicators of salt stress tolerance.

It is important to emphasize that the reduction in fresh weight in all cultivars cannot be attributed solely to the expansion of the leaf area, which plays a key role in the regulation of the transpiration process under osmotic stress. According to several authors [7,33], the reduction in fresh weight is also attributable to a significant and general reduction in leaf initiation (number of leaves). However, the smaller leaf number and area reduction in the Cinnamon × Salt interaction compared to the Anise × Salt and Lemon × Salt ones would elect the Cinnamon cultivar as the most salt tolerant (Figure 3B,C). In our experiment, controlled salt stress induced in plants for 26 days resulted in ionic and nutritional stress, attributed to the accumulation of Na<sup>+</sup> and Cl<sup>−</sup> in transpiration fluxes, leading to a reduction in total fresh weight (Figures 2A and 7).

Although Attia et al. [34] showed that, in basil, Na<sup>+</sup> is partly transported by the xylem and accumulates in the roots, in our work, salt stress resulted in +355% Na<sup>+</sup> accumulation regardless of the cultivar (Figure 7). This result does not exclude the defensive response of the plants to salt since Na+ values did not result in deleterious metabolic dysfunction. In general, the increase in dry matter percentage observed in all cultivars (+35.12%) would suggest that Na+ accumulation was not totally internalized in the leaves, a condition that, as reported by Attia et al. [7], would explain the changes in leaf water content. Although it is frequently documented in the literature [35] that high Na<sup>+</sup> concentrations reduce K, Mg, and Ca uptake, only a reduction in K was observed in our experiment under salt stress; differentially, the concentration of Mg and Ca increased. As Scagel et al. [36] suggested, salt could have reduced Mg and Ca uptake while alternating the allocation of these elements, increasing their concentration. However, since Ca mediates different key processes of adaptation to stress conditions, including the Salt-Overly Sensitive (SOS) pathway, its increase could be related to a long-term response to salinity, as also argued by Mancarella et al. [5]. Similarly to what has been observed for Na+, the use of 60 mM NaCl in the nutrient solution increased the Cl concentration in the leaves, reaching an average value of 36 g kg−<sup>1</sup> dw in the 'Anise' and 'Cinnamon', which explaining the observed worsening production performance (Figures 2 and 3). In fact, the toxicity thresholds for salt-sensitive and salt-tolerant species are 7 and 50 g kg−<sup>1</sup> dw, respectively [37]. As observed in green and red basil, the increase in Cl<sup>−</sup> reduced the nitrate concentration (−77.21%), an antinutritional compound with a negative impact on human health. In salt treatment, the acknowledged antagonism between Cl− and nitrate would have reduced the latter's uptake, slowing the plants' growth rate [38]. Although under salt stress, one of the most common physiological responses is chlorophyll degradation [30], regardless of the cultivar, we did not observe a

reduction in chlorophyll a, b, or total chlorophyll concentrations (Table 2). Consistent with the observations of Bernstein et al. [32], this result suggests that this parameter should be considered an indicator of salt stress for basil. Partially in agreement with the above, the main colorimetric parameters in 'Anise' and 'Cinnamon' under salt stress did not change significantly from what was recorded in the Control (Table 1), considering that leaf color is often correlated with chlorophyll concentration [32]. However, for these two cultivars, salt stress did not significantly alter L, b, chroma, hue angle and especially a\*, referred to in the literature as greenness; these results are probably attributable to the non-significant change in chlorophyll concentration under salt stress.

Unlike in 'Lemon', salt significantly altered colorimetric parameters (Table 1) compared to chlorophyll, and this result could be attributed to significant increases in lutein and β-carotene concentration (Table 2). In addition to their central role as supplementary pigments in photosynthetic processes, carotenoids are crucial for photoprotection due to their antioxidant properties [39]. As Bernstein et al. [32] hypothesized, an increase in carotenoids under salt stress would indicate a protective mechanism against stress, as it is universally recognized that environmental signals control the genetic regulation involved in their biosynthesis and bioaccumulation. Similarly to what was observed for carotenoids, salt stress, regardless of cultivar, increased the concentration of flavonoid derivatives (+35.17) and phenolic acids (+21.54%) and therefore total phenolic compounds (+21.63%; Figure 6). This result is not unexpected, as under NaCl salt stress, the energy stored during photosynthesis is used mainly for growth, with only a part used for synthesizing secondary metabolites. However, mainly by limiting growth, salt would prompt plants to allocate more energy to produce low-carbon secondary metabolites (such as polyphenols) that would help combat ROS [40]. Ghorbanpour and Varma [35] state that the increase in polyphenol concentration in response to salt is related to specific enzymes, including PAL (phenylalanine ammonia lyase). The drastic reduction in osmotic potential, a consequence of salt stress, increases L-proline levels, which is primarily responsible for the osmotic rebalancing. Although this amino acid is a primary metabolite, it may be related to the production of phenolic compounds due to its relationship with PAL [41]. Regardless of salt stress, it is worth noting again how the choice of genetic material significantly affects the bioaccumulation of phenolic compounds [17,42,43]. 'Anise' showed an average concentration of total phenolic compounds 29.12% higher than that recorded in 'Lemon' and 'Cinnamon'. Despite this, the most abundant phenolic acid was chicoric acid, which on average accounted for 63.51% of the phenolic acid derivatives. Although rosmarinic acid is listed in the literature as the most abundant phenolic acid in basil [44], a study of 15 basil cultivars confirmed the influence of genotype on both the quantity and quality of the phenolic profile [45].

'Lemon' was characterized by a 160.57% higher total concentration of flavonoid derivatives compared with 'Cinnamon'. This remarkable difference partially justifies the significant colorimetric differences observed between cultivars, as flavonoids also confer color to vegetables [46]; what has been observed may help to understand the results related to antioxidant activity (Figure 5). Regardless of the methodology used to determine antioxidant activity (DPPH, ABTS, and FRAP), salt increased antioxidant activity, probably due to increased production of compounds with antioxidant activity (carotenoids, phenolic acid derivatives, and flavonoids) [4]. It is well known that phenolic compounds contribute to the antioxidant activity of plant matrices. Therefore, according to Kwee and Niemeyer [45], the different phenolic profile of the three basil cultivars affected the antioxidant power and the activity of DPPH, ABTS, and FRAP (Figure 5). With a 29.50% (on average) concentration of phenolic acid derivatives higher than the other cultivars, 'Anise' showed the highest level of antioxidant activity. On the contrary, although 'Cinnamon' constitutively had a higher concentration of carotenoid (lutein and *β*-carotene) than 'Lemon', it had lower mean values of DPPH, ABTS, and FRAP. This result could be attributable to the higher constitutive concentration of flavonoids in 'Lemon', which by structural characteristics may have positively influenced the antioxidant activities of DPPH, ABTS, and FRAP.
