Introduction

A wide range of abiotic stresses such as high and low temperatures, drought, alkalinity, salinity, UV light, among others, are potentially harmful to plants. Specially, salinity is one of the main factors that adversely affect agricultural production globally, given that more than 800 million hectares worldwide are affected by salinity, representing more than 6% of the planet's surface. Salinity affects crop by causing nutrient imbalance, osmotic stress, water deficiency and oxidative stress (^{Munns and Tester, 2008}).

Salt tolerance in higher plants is a physiologically multifaceted trait, therefore involves different mechanisms. Among the most important osmotic adjustment, reduction in Na ⁺ uptake by the roots and increasing the ion efflux from the cell, intracellular sequestration of Na⁺, accumulation of K⁺ in cytosol; sequestration specific-tissue of Na⁺, charge control of this cation in the xylem, excluding Na⁺ and oxidative stress tolerance (^{Zhu, 2003}).

Particularly, for the tolerance to stress caused by salinity, the plants modified synthesis of secondary metabolites (^{Wahid and Ghazanfar, 2006}). Secondary metabolites are often referred to as compounds not having a key role in maintaining life processes of plants, but they are important for the interaction of the plant with its environment, adaptation and as a defence (^{Ramakrishna and Ravishankar, 2011}).

Thus, in salt tolerant species increased anthocyanin content is a response, when subjected to salt stress (^{Parida and Das, 2005}). Likewise as a consequence of osmotic stress, the osmotic adjustment in the cell cytoplasm to be induced, which leads to the accumulation of low molecular weight osmolytes such as raffinose, betaine glycine and proline, as well as hydrophilic proteins of high molecular weight the superfamily of abundant proteins during late embryogenesis (^{Kosova et al., 2011}). In medicinal plants it has been associated an increase in the concentration of aromatic compounds such as isoprenoids, phenols and alkaloids, as part of its response to salt stress due to the antioxidant properties of these compounds. Furthermore, derivatives of phenylpropanoid phenols, such as flavonoids, tannins and hydroxycinnamates (esters); which are produced under various stress conditions, are major free radical scavengers (^{Selmar, 2008} ).

With respect to the synthesis of essential oils in response to salinity, the results are contradictory. In chamomile (Matricaria Chamomila), irrigated with water with different salinity levels (0, 84, 168, 252 and 336 mM NaCl), the highest content of essential oils in the control treatment (^{Razmjoo et al., 2008}) was recorded. On the other hand, in Tunisian marjoram (Origanum majorana ) a significant increase in the performance of essential oils was recorded when plants were irrigated with water with conductivity of 5 dS m^-1 compared with the control (0 dS m^-1); the increase was 55.5%; while the increase in the irrigation water conductivity to 10 dS m^-1 significantly reduced the yield (^{Jelali et al., 2011}).

Although the effects of salt stress have been extensively investigated in crops, generating information concerning changes of secondary metabolism in medicinal plants such as the cempasúchil is needed in response to salinity. In this context, this study aims to evaluate two levels of salinity from NaCl concentrations of carotenoids and flavonoids in flowers, leaves chlorophyll; as well as the identification and quantification of essential oils for body, in cempasúchil (Tagetes erecta Linn.) var. Inca.

Materials and methods

Experimental conditions and plant material. The research was conducted under greenhouse conditions. Seedlings of cempasúchil (Tagetes erecta Linn.) Var. Inca pots were transplanted into black plastic of 1 litter capacity as a substrate containing a mixture of volcanic rock: perlite (60:40, v: v).

Experiment management. Once transplanted, the seedlings were irrigated with tap water (pH 7.3 and EC 0.37 dS m^-1) for seven days. They were then irrigated with nutrient solution of 25% (^{Steiner, 1984}) supplemented with micronutrients from AZ Tradecorp^™ commercial product at the concentrations described by ^{Trejo-Téllez et al. (2013)}. The pH of the nutrient solution was adjusted to 5.5 and this was supplied by a drip irrigation system. Two irrigations were made each day in every pot, each of which has a volume of 150 mL.

Treatments evaluated. At the beginning of flowering were added to the nutrient solution with NaCl: 10 and 47 mM NaCl, with conductivity values of 1.25Y 5.25%, respectively. Addition of 10 mM of NaCl (1.25 dS m^-1) is not harmful or significantly affected whatsoever in this species (^{Valdez-Aguilar et al., 2009}) therefore can be considered as a reference treatment.

Experimental design. The experimental design was completely randomized, and each treatment had 32 replicates. The experimental unit consisted of a potted plant.

Results and discussion

Total carotenoids. Among the non-enzymatic antioxidants are carotenoids, which are presented ubiquitously in plants and have been reported as vital actors in mitigating adverse effects of salinity on plant growth and metabolism (^{Rawia et al., 2011}). Example of this direct relationship between the level of salinity and concentration of carotenoids are the results of ^{Borghesi et al. (2011)}, who reported that salt stress may lead to similar or higher increases in the fruit of tomato carotenoids and anthocyanins, those made by genetic engineering; therefore, this represents an option with considerable potential exploitation of tomatoes for saline soils with higher levels of secondary metabolites. Also in buckwheat sprouts (Fagorpyrum esculentum M.) treated with 50 and 100 mM NaCl, carotenoid concentration was twice as high in control plants (^{Lim et al., 2012}). Nevertheless, in this study the concentration of total carotenoids in flowers was significantly reduced with the addition of 47 mM NaCl, compared to the supply of 10 mM NaCl; a reduction of 26.7% (Figure 1).

Figure 1 Total carotenoids in cempasúchil flowers var. Inca, treated with two concentrations of NaCl in flowering stage. Mean ± SD with different letters indicate significant differences (Tukey, p≤ 0.05) between treatments. 

Total flavonoids. The concentration of flavonoids in flowers had a negative relationship with the concentration of NaCl in the nutrient solution; observing an increasing of NaCl from 10 to 47 mm 27.9% reduced in their concentration (Figure 2). Similarly, in chamomile (Matricaria chamomilla L.) treated with varying levels of NaCl (40, 80, 120 and 190 mM), there was a reduction in the concentrations of flavonoids than the control’s (without NaCl); however, with a NaCl concentration of 40 mM, we found the highest content of flavonols 3 -O-glycosides, flavonoids most abundant flowers of this species (^{Afzali et al., 2007}).

Figure 2 Total flavonoids in flowers of cempasúchil var. Inca, treated with two concentrations of NaCl in flowering stage. Mean ± SD with different letters indicate significant differences (Tukey, p≤ 0.05) between treatments. 

Opposite to the results of flavonoids obtained in this study, in hypocotyls and cotyledons of seedlings of tomato (Solanum esculentum cv. H-2274) and red cabbage (Brassica oleraceae convar. capitata (L.) Alef. Var. Rubra DC. Cv Möhrenkopf), gradual increases were observed in the concentrations of anthocyanins, as the NaCl concentration increased from 0 to 150 mM (Eryılmaz, 2006).

Total chlorophylls. The Table 1 shows the leaf chlorophyll concentrations under two levels of NaCl. Total concentrations of chlorophyll a and were not affected by the level of NaCl added to the nutrient solution; while the concentration of chlorophyll b was statistically superior 47 mM NaCl, 10 mM compared.

Studies on the effects of salt stress in plants have mainly focused on growth, proline accumulation, chlorophyll content, relative K⁺/Na⁺, Ca2 ⁺ ratio /Na⁺, accumulation of Na ⁺ and Cl^-. It has been said that genotypes with a high accumulation of proline and chlorophyll contents, as well as high ratio K⁺/Na⁺ and low accumulation of Na⁺ and Cl^- are more tolerant (^{Mane et al., 2011}). Considering the above, we can say that Tagetes erecta var. Inca is a tolerant species at levels of 47 mM NaCl.

Essential oils. The retention times of the essential oil compounds identified in leaves, flowers, stems and roots are presented in Table 2.

Is indeed quite important to highlight the large difference in the components of essential oils from Tagetes species, since the compounds identified here do not match those reported in Tagetes coronopifolia Willd, which are in roots 2, 7, 7-trimetilbiciclo[3.1.1] heptan-2-ol y (1S, 2R, 5S) -4, 6,6-trimetilbiciclo[3.1.1] hept-3-en-2-ol (verbenol) (38%); (1S) -6,6-dimetil-2-metilen-biciclo[3.1.1] heptan-3-ona, (1R)-cis-4,6,6-trimetilbiciclo-[3.1.1] hept-3-en-2-ona (verbenona), 2-oxo-decanoato de metilo and 2,7,7-trimetilbiciclo[3.1.1] hept-2-en-6-ona (crisantenona) (41%); while in the flowers, (1S)-6,6-dimetil-2-metilen-biciclo[3.1.1] heptan-3-ona and verbenona (^{Díaz-Cedillo et al., 2013}).

The number of compounds and their concentration in the oil from leaves were higher with low salinity level tested (10 mM), the most abundant D-limonene (8.52%) and ocimene (6.33%) and higher at 58.92 and 46.13%, respectively, for the content thereof recorded in leaves of plants treated with 47 mM NaCl. It is also important to note that only by the addition of 10 mM NaCl, trans-pinene, β-myrcene and piperitone (Figure 3A) were identified. ^{Rawia et al. (2011)} reported oil from fresh tissue of Tagetes erecta plants subjected to three concentrations of NaCl (salt), 10 compounds: cis-β-ocimene, β-farnesene, trans-linalool oxide, piperetona, β-ionones, trans-cariofilasa oxide cariofilasa , eugenol, and hexa-decanoice linallool acid methyl ester; in all these, the relative concentration was reduced as the concentration of NaCl is increased. Regardless of the treatment, the most abundant compounds were trans-caryophyllene and eugenol. Therefore, this study overlaps with the results of this research; lower relative concentration of essential oils increases as the concentration of NaCl; and differ in the amount and type of identified compounds.

Figure 3 Essential oils identified in leaves (A), flowers (B), stems (C) and roots (D) of cempasúchil var. Inca, treated with two concentrations of NaCl in flowering stage. 

Contrary to the results of the leaves, in the flowers were found three compounds in essential oils with higher salinity; while with lower salinity only two, caryophyllene and caryophyllene oxide. Farnesene was identified only under higher NaCl concentration of 1.28 percent (Figure 3B). Coincidentally, in peppermint plants (Mentha piperita L.), increases were observed in some essential oil components in response to NaCl treatment, including the trans-β-farnesene is located; with relative concentrations of 0.01 and 0.89%, with the treatment 0 and 50 mM of NaCl, respectively; while with 100 mM NaCl was not identified. In the case of the relative concentration of L-menthol, a major constituent in the essential oil of the species is gradually increased with increasing NaCl dose of 50 and 100 mM (^{Khorasaninejad et al., 2010}).

Similar to those recorded in flower trends are observed in stems; i.e. three compounds identified in plants treated with 47 mM; while only piperitone was detected with the lowest concentration of 10 mM NaCl; however, its concentration exceeded that found with higher concentrations of NaCl in 26.63% (Figure 3C).

In roots, the high dose of NaCl were caryophyllene, farnesene and caryophyllene oxide identified; of these one-farnesene with increasing NaCl. Just the piperitone compound was identified in roots of plants subjected to low salinity levels (Figure 3D).

Consistently, we observe that the relative concentration of piperitone in oil from leaves, stems and roots was significantly affected by increased salinity (Figure 3).

The increase in essential oil content, as seen in flowers, stems and roots of this species has been reported in other, subject to factors that cause osmotic stress. The content of essential oil and basil proline were increased when plants are subjected to drought stress; the growth was inhibited significantly (^{Baeck et al., 2001}). In addition, ^{Hendawy and Khalid (2005)} reported that, the essential oil content, total carbohydrates and proline were increased significantly with the increase in the level of salt stress in plants of salvia (Saliva officinalis L. Sage).

Conclusions

The concentrations of carotenoids and flavonoids were adversely affected while increasing NaCl; in contrast, the chlorophyll a and total concentrations were not affected by the level of NaCl, while the concentration of chlorophyll b was increased with increasing NaCl concentration.

The treatment with 47 mM NaCl reduces the essential oil components of leaves and reduces the relative concentrationsas well. In flowers, stems and roots; in contrast, with the increasing level of NaCl, generally essential oil components and their concentrations were also increased, noting the trend in stems and roots.