An assessment of the physicochemical characteristics and essential oil composition of Mentha longifolia (L.) Huds. exposed to different salt stress conditions

Abstract

Salt stress adversely influences growth, development, and productivity in plants, resulting in a limitation on agriculture production worldwide. Therefore, this study aimed to investigate the effect of four different salts, i.e., NaCl, KCl, MgSO₄, and CaCl₂, applied at various concentrations of 0, 12.5, 25, 50, and 100 mM on the physico-chemical properties and essential oil composition of M. longifolia. After 45 days of transplantation, the plants were irrigated at different salinities at 4-day intervals for 60 days. The resulting data revealed a significant reduction in plant height, number of branches, biomass, chlorophyll content, and relative water content with rising concentrations of NaCl, KCl, and CaCl₂. However, MgSO₄ poses fewer toxic effects than other salts. Proline concentration, electrolyte leakage, and DPPH inhibition (%) increase with increasing salt concentrations. At lower-level salt conditions, we had a higher essential oil yield, and GC-MS analysis reported 36 compounds in which (-)-carvone and D-limonene covered the most area by 22%-50% and 45%-74%, respectively. The expression analyzed by qRT-PCR of synthetic Limonene (LS) and Carvone (ISPD) synthetic genes has synergistic and antagonistic relationships in response to salt treatments. To conclude, it can be said that lower levels of salt enhanced the production of essential oil in M. longifolia, which may provide future benefits commercially and medicinally. In addition to this, salt stress also resulted in the emergence of novel compounds in essential oils, for which future strategies are needed to identify the importance of these compounds in M. longifolia.

Keywords: biochemical; essential oil; growth; metabolites; physicochemical; salt stress.

Figure 1

Meteorological data on maximum and minimum temperature (°C), maximum and minimum relative humidity (%) and total rainfall (in) during the experiment (from plant transplantation to harvesting). TM, temperature; RH, relative humidity; RF, rainfall.

Figure 2

Morphology of M. longifolia plants (A—shoot and B—root) treated with salts. (1) NaCl, (2) KCl, (3) MgSO₄, and (4) CaCl₂.

Figure 3

Effect of 12.5, 25, 50, and 100 mM concentrations of NaCl, KCl, MgSO₄, and CaCl₂ salts on leaf relative water content (%) in an M. longifolia leaf sample. The data are represented as mean ± standard deviation (n = 3). The different letters on the bar graph show the significantly different groups according to the Duncan test (α = 0.05).

Figure 4

Effect of 12.5, 25, 50, and 100 mM concentrations of NaCl, KCl, MgSO₄, and CaCl₂ salts on electrolyte leakage (%) in an M. longifolia leaf sample. The data are represented as mean ± standard deviation (n = 3). The different letters on the bar graph show the significantly different groups according to the Duncan test (α = 0.05).

Figure 5

1. Chlorophyll content and total carotenoid content (mg g⁻¹) in an M. longifolia leaf sample under salt stress condition. (A) Different concentrations of NaCl; (B) Different concentrations of KCl; (C) Different concentrations of MgSO₄; and (D) Different concentrations of CaCl₂. The data are represented as mean ± standard deviation (n = 3). The different letters on the same color bar show the significantly different groups according to the Duncan test (α = 0.05). 2. Photosynthetic pigment in an M. longifolia leaf sample under different salt stress conditions.

Figure 6

Effect of 12.5, 25, 50, and 100 mM concentrations of NaCl, KCl, MgSO₄, and CaCl₂ salts on proline content (µg g⁻¹ fresh weight) in an M. longifolia leaf sample. The data are represented as mean ± standard deviation (n = 3). The different letters on the same color bar show the significantly different groups according to the Duncan test (α = 0.05).

Figure 7

Effect of 12.5, 25, 50, and 100 mM concentrations of NaCl, KCl, MgSO₄, and CaCl₂ salts on DPPH inhibition % (antioxidant activity) in an M. longifolia leaf sample. The data are represented as mean ± standard deviation (n = 3). The different letters on the same color bar show the significantly different groups according to the Duncan test (α = 0.05).

Figure 8

Effect of 12.5, 25, 50, and 100 mM concentrations of NaCl, KCl, MgSO₄, and CaCl₂ salts on essential oil yield in an M. longifolia leaf sample.

Figure 9

GC–MS chromatograms of the dominant compounds (A) limonene and (B) carvone of essential oil in M. longifolia.

Figure 10

Limonene % area obtained from GC–MS analysis in an M. longifolia essential oil sample under salt stress condition. (A) Different concentrations of NaCl; (B) Different concentrations of KCl; (C) Different concentrations of MgSO₄; and (D) Different concentrations of CaCl₂. The data are represented as mean ± standard deviation (n = 3).

Figure 11

Carvone % area obtained from GC–MS analysis in an M. longifolia essential oil sample under salt stress condition. (A) Different concentrations of NaCl; (B) Different concentrations of KCl; (C) Different concentrations of MgSO₄; and (D) Different concentrations of CaCl₂. The data are represented as mean ± standard deviation (n = 3).

Figure 12

Correlation among chlorophyll (a), chlorophyll (b), chlorophyll (a + b), total carotenoids, leaf relative water content, electrolyte leakage, proline content, DPPH inhibition %, carvone and limonene % area. The brown color boxes show a strong correlation, and the purple color boxes show a weak correlation.

Figure 13

Principal component analysis (PCA) biplots of M. longifolia chlorophyll (a), chlorophyll (b), chlorophyll (a + b), total carotenoids, leaf relative water content, electrolyte leakage, proline content, DPPH inhibition %, carvone and limonene % area with different (12.5, 25, 50, and 100 mM) concentrations of NaCl, KCl, MgSO₄, and CaCl₂ salts compared to the control plant.

Figure 14

qRT-PCR-based expression analysis of key genes of the carvone and limonene biosynthesis pathways of M. longifolia. The bar diagrams represent the fold change in expression of each gene upon 12.5, 25, 50, and 100 mM salt treatment as compared to untreated controls. (A–D) Different concentrations of NaCl; (E–H) Different concentrations of KCl; (I–L) Different concentrations of MgSO₄; and (M–P) Different concentrations of CaCl₂. The results are presented as the means of three replicates and as means ± SDs. Statistical significance was determined by the Student’s t-test. Letters a and b denote the significance of fold changes at p-values <0.05 (a <0.005, b<0.05) while letter c denotes the non-significant nature of fold changes at p-values >0.05.