Effects of Temperature, pH, and Salinity on Seed Germination of Acinos alpinus subsp. Meridionalis and FTIR Analysis of Molecular Composition Changes

Abstract

This study aimed to determine the impact of three abiotic factors (Temperature, pH, and salinity) on the metabolic activities (macromolecules) and germination rate of Acinos alpinus subsp. Meridionalis (Satureja) seeds. In vitro, seed germination tests were performed in an aqueous medium. They were monitored as a function of time at different levels of temperature, NaCl concentration, and pH. The best germination rate (85.3%) was achieved at 15 °C and pH = 7. However, the germination was nil at a higher temperature (more than 25 °C), acidic pH (pH < 3.5), and higher NaCl concentration (more than >7.5 g L⁻¹). Fourier transform infrared spectroscopy (FTIR) analysis showed an important variability of the chemical composition of germinated seeds. Indeed, the comparison of the absorbance peaks of chemical compounds in the treatments versus the control revealed significant differences in their concentrations and structures, which may justify why seeds fail to germinate under some extreme abiotic conditions. The results of this study are expected to serve as a guide for the protocols to be adopted in the ex situ conservation of this species.

1. Introduction

One of the most important medicinal species according to ethnobotanical studies are those that belong to the Acinos sp. They are used in several countries for their aromatic, medicinal, and culinary properties [1,2]. Those species are used as antispasmodics, antiseptics, tonics, diuretics, antipyretics, and stimulants in the treatment of diarrhea, indigestion, obesity, respiratory diseases, cough, melancholy, toothache, sciatica, and neuralgia, especially in Mediterranean countries [3,4,5].

They belong to the Lamiaceae family. The current data indicate that the genus Acinos includes 11 species with many synonyms and some species are endemic to the Mediterranean region [2,5]. Among those species, we have Acinos alpinus (L.) Moench or Satureja alpina (L.) Scheele, which is locally known as common thyme, “Tamtot Flio”, “Saitra”, “Saatr”, and “Flio Bori”. Its leaves are not curled at the edges and have veins that aren’t projecting below. It has deep violet or purple corolla, 1–2 cm long. The upper lip is flat and indented, while the lower lip’s median lobe is relatively large. The flowers outnumber the bracts. After flowering, the teeth of the calyx do not close, and the three top ones are noticeably shorter than the two lower ones (Figure 1). It is a cedar forest species that has a basic predisposition to grow on limestone or dolomitic substratum from the Lias and Jurassic periods in subhumid and humid stages at altitudes ranging from 1500 to 2000 m, where the yearly precipitations reach 1000 mm [6]. They disseminate their seeds in late summer or the early fall in their natural habitat, then they germinate once endogenous dormancy is broken. Here, it is worth mentioning that the time of germination depends on the area. In semi-arid climates, they germinate in May, and in humid climates, they germinate through June and July [3]. Moreover, abiotic and biotic factors have a marked impact on seed germination [7,8,9]. They can delay, reduce, and also prevent germination [10,11]. For instance, salinity is a factor that not only limits plant growth but also seed germination (Koyro 2006). It reduces the plant’s capacity for water absorption, which triggers several physiological and metabolic processes causing the prolongation of seed germination time and this mainly happens by increasing the osmotic pressure [12]. Furthermore, Guan et al. [13] reported that seed germination periodicity is mostly affected by temperature. The germination rate increases with temperature proportionally up to its optimum and then it decreases sharply [14,15]. Likewise, soil pH is also reported as one of the factors that significantly influence seed germination [9,16].

There are studies [17,18,19] that have worked on the effect of abiotic factors on the germination of some species of Lamiaceae; however, few works [16,20] take into account the same factors that we have tried. To the best of our knowledge, there is no research reporting the effect of abiotic stresses on A. alpinus seeds. The main objective of this paper is to fill such a gap by investigating changes caused by salinity, pH, and temperature in the germination rates and metabolic activities (macromolecule levels in germinated seeds) of this species. Furthermore, it aims to establish optimal conditions for A. alpinus seed germination, which would contribute greatly to reducing the risk of extinction due to overexploitation and environmental stresses [21].

2. Material and Methods

2.1. Seeds Collection

Plant samples were collected from Ain Leuh (Ifrane Province, northern Morocco) in June 2018 (Figure 2). Thereafter, the samples were dried at room temperature until constant weight. Then, the mature seeds were extracted manually and delicately from the blossoms. The size and weight of the extracted seeds were 1.42 ± 0.23 mm and 0.18 mg, respectively. Those seeds were distributed in pods (three to four seeds per pod).

2.2. Germination Experiments

Before each germination experiment, the seeds were placed in a sodium hypochlorite solution (5%) for 5 min to prevent fungal development [18]. Thereafter, the seeds were rinsed with sterile distilled water (SDW) and air-dried at room temperature (25 °C). Then, the seeds were placed in Petri dishes on Whatman filter paper, which were moistened with 1.5 mL of SDW [16].

Three different germination experiments were carried out. In the first one, five different temperature levels were tested: 5, 10, 15, 20, and 25 °C. To determine the salt stress impact, seven levels of NaCl were applied: 0, 1, 2.5, 5, 7, and 10 g L⁻¹. For the evaluation of the pH effect, the germination test was conducted at different pHs (2, 3.5, 5, 7, 9, 11, and 12.5). The NaCl and pH experiments were performed under the optimal temperature defined previously (15 °C). For all three experiments, the Petri dishes were closed with parafilm and incubated in the oven to ensure dark conditions. A seed was considered germinated once it presented, at least, a 1 mm long radical [22]. For each Petri dish, the cumulative germination rate was calculated daily [23]. For all the experiments, three replicates (Petri dishes) of 25 seeds/Petri dish were considered.

2.3. FTIR Spectroscopy Analysis

Seeds that failed to germinate in different experimental treatments and seeds that did not receive any treatment (control) were ground and directly placed upon the diamond ATR crystal (Spectrum Two FTIR Spectrometer PerkinElmer, Waltham, MA, USA) and pressed by the ATR accessory. Attenuated total reflectance spectra were scanned in the wavelength range of 8300 to 350 cm⁻¹ with a resolution of 4 cm⁻¹ and a signal-to-noise ratio peak-to-peak of 9.300:1 for 5 s of acquisition. The spectra were taken in triplicate then the average was calculated from the three measures. Before each ATR measurement, the background spectrum was fulfilled. Spectral data were collected through the compressing of all samples using the accessory ATR. Ethanol was used to clean the ATR cell after each use to avoid sample contamination [24,25].

2.4. Statistical Analysis

The main effects of temperature, salt stress, and pH on germination rates were evaluated using one-way ANOVA, followed by Duncan’s multiple-range mean separation test. Normality was verified using the Shapiro–Wilk test, and equality of variance was verified using Levene’s test. The Mann–Whitney U test (two-tailed) or two independent t-tests were used to determine if the absorbance peaks of macromolecules characterized by FTIR analysis in the treatment groups differed significantly from those in the control group. The choice of test was based on the normality of the data, as determined by the Shapiro–Wilk test. The significance level was set at p < 0.05, and the statistical analyses were conducted using SPSS software (Version 21, IBM SPSS Statistics for Windows).

3. Results

3.1. Effects of Temperature, Salinity, and pH on Seed Germination

A clear tendency appeared between the different temperature levels from the early days of the test (Figure 3a). At the end of the test, the germination rate was significantly affected by the temperature (Figure 3b). The rapid and highest germination rate (85.3%) was observed at 15 °C. However, no seeds germinated at 5 and 25 °C. The germination evolution curve showed that a latency time exists between the first and the third day. At 10 and 20 °C, the latency period lasted 3 days and the germination rate fluctuated between 40% and 48%, respectively.

Figure 3c shows the effect of NaCl on seed germination for 28 days of cultivation. The germination percentage was at its maximum (85.3%) in the control (0 g L⁻¹) and the latency period was extremely short, lasting one day. Based on the variance analysis, the germination percentage was affected significantly (p < 0.05) by the salinity stress (Figure 3d). The exponential germination phase lasted around ten days before reaching the stationary phase and stopped at the maximum germination percentage. A negative correlation (R² = 0.9435; p < 0.05) was obtained between germination rate and NaCl concentrations, indicating that if the salinity increased, it would reduce the A. alpinus seeds’ germination.

The effect of pH on seed germination of A. alpinus is shown in Figure 3e. Seed germination took place in a range of pH between 3.5 and 11. Under low pH (pH = 2), no seeds germinated. However, at pH = 7, the maximum (85.3%) germination was reported (Figure 3f), which qualified as optimum germination pH. The germination rate decreased when the pH exceeded its optimum. Above pH = 11, the seeds of A. alpinus were not able to germinate.

3.2. Characterization of A. alpinus Seeds’ Macromolecules Using ATR-FTIR Spectra

The ATR-FTIR spectrum analyses were carried out on the different seeds undergoing extreme abiotic factors (pH, salinity, and temperature) whose seeds could not germinate. Results show significant differences in absorbance intensities (Figure 4). Untreated seeds are considered as references since they germinate normally with a very high rate of 85.3%. According to the results from the FTIR analyses, it seems that the seeds subjected to different stresses of temperature, acidity, and salinity show different curves than the control seeds (

Table 1. Changes in the peaks and peak ratios in control and different treatments (n = 3). Data are shown as mean ± SD. Differences between the treatment groups and the control group were analyzed by Mann–Whitney U or Student’s test.; (a): p value with the use of two independent t-tests; (b): p-value with the use of the Mann–Whitney U test; ν, stretching; δ, bending; w, wagging; as, asymmetric; s, symmetric.

		Control		T = 5 °C		T = 25 °C		pH = 2		pH = 12.5		NaCl = 10
Peaks and Ratios	Assignment	References	Mean (±SD)	Mean (±SD)	p-Value	Mean (±SD)	p-Value	Mean (±SD)	p-Value	Mean (±SD)	p-Value	Mean (±SD)	p-Value
3283	N-H, O-H	[26]	0.034 ± 0.004	0.03 ± 0.001	0.3 (a)	0.052 ± 0.002	0.002 (a)	0.013 ± 0.001	0.013 (a)	0.031 ± 0.001	0.37 (a)	0.05 ± 0.003	0.006 (a)
3010	ν = CH (unsaturated lipids)	[27]	0.04 ± 0.002	0.019	0.1 (b)	0.036 0.001	0.1 (b)	0.017 ± 0.001	0.1 (b)	0.029	0.1 (b)	0.041 ± 0.001	0.4 (b)
2960	νasCH3	[28]	0.047 ± 0.003	0.02	0.1 (b)	0.04 ± 0.001	0.1 (b)	0.018 ± 0.002	0.1 (b)	0.033	0.1 (b)	0.048 ± 0.001	0.4 (b)
2925	νasCH2 (lipids)	[29]	0.092 ± 0.003	0.037	0.001 (a)	0.037	0.1 (b)	0.039 ± 0.004	p < 0.001 (a)	0.067 ± 0.001	0.005 (a)	0.09 ± 0.001	0.434 (a)
2874	νsCH3 (lipids)	[28]	0.04 ± 0.002	0.022	0.1 (b)	0.038 ± 0.001	0.4 (b)	0.018 ± 0.001	0.1 (b)	0.031	0.1 (b)	0.043 ± 0.001	0.1 (b)
2853	νsCH2 (lipids)	[30]	0.063 ± 0.002	0.028	0.001 (a)	0.052	0.1 (b)	0.028 ± 0.002	0.1 (b)	0.048	0.006 (a)	0.062	0.557 (a)
1745	ν(C = O) (pectin, polysaccharide)	[31]	0.067 ± 0.001	0.024	0.1 (b)	0.044 ± 0.002	p < 0.001 (a)	0.031 ± 0.003	0.1 (b)	0.045 ± 0.001	p < 0.001 (a)	0.06	0.001 (a)
1650	Amide I	[32]	0.031 ± 0.004	0.014	0.014 (a)	0.031 ± 0.001	0.988 (a)	0.009	0.1 (b)	0.017	0.1 (b)	0.033 ± 0.001	0.323 (a)
1540	Amide II	[33]	0.018 ± 0.002	0.007	0.014 (a)	0.018 ± 0.002	0.927 (a)	0.004	0.01 (a)	0.01	0.1 (b)	0.017 ± 0.001	0.601 (a)
1517	Amide II	[28]	0.015 ± 0.002	0.008	0.024 (a)	0.017 ± 0.002	0.376 (a)	0.004	0.011 (a)	0.009	0.1 (b)	0.016 ± 0.001	0.603 (a)
1460	δ[(C-H] (lipids)	[34]	0.022 ± 0.001	0.011	0.002 (a)	0.021 ± 0.002	0.7 (b)	0.01 ± 0.001	0.1 (b)	0.017	0.1 (b)	0.021	0.7 (b)
1455	δ[(CH3)]as	[35]	0.022 ± 0.001	0.012	0.003 (a)	0.021 ± 0.002	0.7 (b)	0.01 ± 0.001	0.1 (b)	0.017	0.013 (a)	0.022	0.921 (a)
1375	δ(C-H)	[36]	0.015 ± 0.001	0.011	0.1 (b)	0.017 ± 0.002	0.1 (b)	0.008	0.1 (b)	0.014	0.1 (b)	0.018	0.1 (b)
1315	δs(CH2)w	[37]	0.013 ± 0.001	0.009	0.1 (b)	0.015 ± 0.002	0.1 (b)	0.007	0.1 (b)	0.012	0.1 (b)	0.014	0.1 (b)
1271	δ(OH) (cutin and polysaccharides)	[38]	0.015 ± 0.001	0.01	p < 0.001 (a)	0.015 ± 0.003	0.7 (b)	0.008 ± 0.001	p < 0.001 (a)	0.011	0.1 (b)	0.016	0.1 (b)
1238	[PO2-(as)] (nucleic acids and phospholipids)	[39]	0.02 ± 0.001	0.014	p < 0.001 (a)	0.02 ± 0.003	0.967 (a)	0.011 ± 0.001	p < 0.001 (a)	0.014	p < 0.001 (a)	0.023	0.003 (a)
1159	ν(C-O) (carbohydrates, proteins)	[30]	0.033 ± 0.001	0.015	p < 0.001 (a)	0.029 ± 0.003	0.1 (b)	0.017 ± 0.001	p < 0.001 (a)	0.026	p < 0.001 (a)	0.034	0.137 (a)
1100	Carbohydrates	[40]	0.024 ± 0.001	0.014	0.003 (a)	0.027 ± 0.002	0.1 (b)	0.011 ± 0.001	p < 0.001 (a)	0.02	0.1 (b)	0.03 ± 0.001	0.002 (a)
1066	ν(C-O) (rhamnogalacturonan/pectin)	[41]	0.023 ± 0.001	0.017	0.003 (a)	0.03 ± 0.002	0.007 (a)	0.011 ± 0.001	p < 0.001 (a)	0.019	0.05 (a)	0.033 ± 0.001	p < 0.001 (a)
1060	ν(C-O) (D-ribose)	[35]	0.022 ± 0.001	0.019	0.022 (a)	0.032 ± 0.002	0.002 (a)	0.011 ± 0.001	0.1 (b)	0.02	0.1 (b)	0.036 ± 0.001	p < 0.001 (a)
1049	ν(C-O) (D-ribose)	-	0.02 ± 0.001	0.02	0.97 (a)	0.032 ± 0.002	0.001 (a)	0.01 ± 0.001	0.1 (b)	0.019	0.4 (b)	0.037 ± 0.001	p < 0.001 (a)
1031	ν(CC), ν(CH2OH), ν(CO)	[42]	0.019 ± 0.001	0.021	0.07 (a)	0.032 ± 0.002	p < 0.001 (a)	0.01 ± 0.001	0.1 (b)	0.019	0.7 (b)	0.038 ± 0.001	p < 0.001 (a)
899	ν(COC), C-C out of plane bending	[43]	0.005	0.007	p < 0.001 (a)	0.007 ± 0.001	0.1 (b)	0.005	0.002 (a)	0.006	0.1 (b)	0.007	p < 0.001 (a)
2925/2960	νasCH2/νasCH3	-	1.977 ± 0.035	1.793 ± 0.011	0.001 (a)	1.786 ± 0.023	0.001 (a)	2.119 ± 0.002	0.019 (a)	2.006 ± 0.013	0.245 (a)	1.877 ± 0.017	0.011 (a)
2853/2874	νsCH2/νsCH3	-	1.596 ± 0.028	1.307 ± 0.01	p < 0.001 (a)	1.358 ± 0.015	p < 0.001 (a)	1.571 ± 0.019	0.274 (a)	1.527 ± 0.01	0.016 (a)	1.452 ± 0.015	0.001 (a)
2853/2960	νsCH2/νasCH3	-	1.357 ± 0.029	1.395 ± 0.007	0.088 (a)	1.285 ± 0.012	0.016 (a)	1.53 ± 0.009	0.001 (a)	1.437 ± 0.008	0.1 (b)	1.297 ± 0.014	0.1 (b)
2925/2874	νasCH2/νsCH3	-	2.326 ± 0.032	1.681 ± 0.015	p < 0.001 (a)	1.888 ± 0.028	p < 0.001 (a)	2.176 ± 0.04	0.007 (a)	2.132 ± 0.016	0.001 (a)	2.101 ± 0.017	p < 0.001 (a)
1650/2853	Amide I/νsCH2	-	0.332 ± 0.027	0.388 ± 0.006	0.023 (a)	0.428 ± 0.016	0.006 (a)	0.233 ± 0.014	0.005 (a)	0.261 ± 0.007	0.011 (a)	0.367 ± 0.012	0.109 (a)
1650/1540	Amide I/Amide II	-	2.036 ± 0.036	1.772 ± 0.012	p < 0.001 (a)	1.854 ± 0.136	0.1 (b)	2.061 ± 0.052	0.7 (b)	2.035 ± 0.001	0.7 (b)	2.102 ± 0.008	0.084 (a)
3010/(2925 + 2853)	Unsaturation index (U.I)	-	0.258 ± 0.005	0.298 ± 0.002	0.1 (b)	0.289 ± 0.004	0.1 (b)	0.253 ± 0.005	0.2 (b)	0.249 ± 0.001	0.1 (b)	0.272 ± 0.003	0.1 (b)

).

At T = 5 °C, there is a statistically significant (<0.05) decrease in intensities of 2925, 2853, 1650, 1540, 1517,1460, 1455, 1271, 1238, 1159, 1100, 1066, 1060, and 899 cm⁻¹ as well as the ratio of 2925/2960, 2853/2874, 2925/2874, 1650/2853, and 1650/1540.

At T = 25 °C, there is a statistically significant decrease in intensities of 1745 cm⁻¹ as well as the ratio of 2925/2960, 2853/2874, 2853/2960, and 2925/2874. However, there is an increase in intensities of 3283, 1066, 1060, 1049, and 1031 cm⁻¹ and a ratio of 1650/2853.

At pH = 2, there is a statistically significant decrease in intensities of 3283, 2925, 1271, 1238, 1159, 1100, 1066, and 899 cm⁻¹ and ratios of 2925/2874 and 1650/2853. However, there is an increase in the ratio of 2925/2960 and 2853/2960.

At pH = 12.5, there is a decrease of 2925, 2853, 1745, 1238, and 1159 and a ratio of 2853/2874, 2925/2874, and 1650/2853.

At NaCl =10 g L⁻¹, there is a significant decrease in intensities of 1745 and ratios of 2925/2960, 2853/2874, and 2925/2874. However, there is an increase in intensities of 3283, 1100, 1066, 1060, 1049, and 1031 cm⁻¹.

4. Discussion

4.1. Influence of Temperature, Salinity, and pH on Germination

The study of temperature effect on seed germination showed an important germination rate (85.3%) at 15 °C with a latency time of 1 to 3 days. This optimum germination at 15 °C, which is similar to the bio-ecological environment of the collection site between April and May, confirmed that the performance reflects the adaptation of the plant to its ecological origin as has been stated by Sento [44]. Furthermore, the results at 10 and 20 °C showed that the latency period lasted 2 to 3 days and the germination percentage fluctuated between 40% and 48%, respectively. However, we found complete germination inhibition at 5 and 25 °C. These results could be explained by the fact that temperature has a direct impact on metabolism and germination speed, which are a series of biological reactions involving enzymes that can only be active within a specific temperature range. For instance, at a high temperature (25 °C), germination of A. alpinus seeds was significantly inhibited. This thermoinhibition could be attributed to the rising doses of endogenous abscisic acid (ABA) to which seeds are very sensitive [45].

Besides its crucial role in catalyst activation, temperature plays a role in gibberellic acid (GA) accumulation by stimulation of transcription factors via different chromatin channels [46]. Studies reported that the optimal germination temperature can vary from one species to another or even from one plot to another for the same species; Eberle et al. (2014) [47] reported that the germination optimal value for Calendula arvensis was around 15 °C. However, Fallahi et al. [14] demonstrated that the temperature ranged between 25 and 30 °C for green and purple basil, respectively. These differences can be explained by environmental factors and genetic specificities [16,20].

Salt stress negatively affected the germination rate of the studied species, as is the case of seeds from most species [10,11]. By increasing salt stress, the exponential phase shape delays at a slower rate. The germination inhibition by NaCl could be shown either as complete inhibition when salinity exceeds 7 g L⁻¹, which is the tolerance limit, or as delaying seed germination at stressing salinity levels of 5 g L⁻¹ where the seeds show a less developed and yellowish outline signifying the stress undergone by the gemmule. Hence, this species appears to be less tolerant of saline stress during the germination period than numerous other spontaneous plants, such as Origanum compactum [16].

The high negative correlation observed between the germination rate of A. alpinus and NaCl concentration revealed that any increase in salinity will result in seed germination reduction. However, the linear correlation was not reported in situ as it was in vitro. This result shows how difficult it is to link salinity tolerance at germination time to the species’ ecology or tolerance at the mature plant stage. The search for a correlation between salinity and germination rate in the soil must take into account additional interconnected factors [16].

The decrease in seed germination at high salinity could be a marker of a dormant osmotic process, which was a response to salt stress conditions, which represented a strategy of managing environmental issues by plants [8] and also of the changes that occur by several hormone and enzyme functions involved in this process [48,49]. The high NaCl levels significantly decreased the germination rate, which can be due to a decrease in water uptake created by salinity conditions and/or the toxicity effects of Na⁺ and Cl^- ions on the germination process [50]. Excess ions can cause many disorders in the metabolic processes of germination, and, in the worst-case scenario, the embryo dies [51]. In addition, a high osmotic potential caused by salt stress could lead to cell dehydration, which negatively affects germination [52]. It is worth mentioning also that when the concentration of NaCl increased (over 1 g L⁻¹), the lag period was lengthened. These findings are consistent with those of a study on the Thymus marroccanus Ball. germinating seeds, which found latency in germination [53]. The time it takes for the seeds to activate the systems that allow them to regulate their osmotic pressure could explain the prolonging of the lag period [54]. Botía et al. [48] attributed the delay to changes in enzymes and hormones in the seed. Other studies imply that NaCl impacts germination in two ways: by completely inhibiting it with levels that exceed tolerance limits, or by prolonging it by seed stress [55,56].

pH significantly affected the germination percentage. The germination rate reached a maximum value of 85.3% at pH = 7. At low and high pH values (pH = 2 and 12.5), seeds of A. alpinus failed to germinate. The synthesis and the activity of the enzymes involved in the germination process are inhibited in hyper acidic environments that also affect the seed integuments by dissolving it or by stimulating several pathogens’ development, causing its perforation [9]. Moreover, the time seeds take to germinate could be postponed in hyper alkaline environments [20]. Although high pH did not interfere with seed water absorption when alkalinity was low, alkaline salts severely harmed the lateral root that broke through the seed coat, causing seeds to germinate but struggle to form regular seedlings that could survive [57].

4.2. Characterization of A. alpinus Seeds’ Macromolecules Using ATR-FTIR Spectra

ATR-FTIR is a technique that is effectively used for the determination of fingerprint properties that are unique for samples. Our results strengthen the fact that environmental stresses affect seed metabolic activities. In fact, at different stresses, FTIR analysis shows significant differences between the treatments and the control in the ratio of 2925/2960, 2853/2874, 2853/2960, and 2925/2874 indicating a dramatic change in chain length and branching of seeds lipids under the studied abiotic stresses (pH, temperature, and salinity) and a significant decrease in 1238 cm⁻¹, indicating a decrease in nucleic acids and phospholipids under these stresses. The statistically significant changes in the peak 1100 cm⁻¹ indicate an increase in seed carbohydrate content in hypersaline environments and its decrease in acidic and cold conditions.

In the extreme temperatures (5 and 25 °C) where seeds failed to germinate, FTIR analysis shows an increase in 1650/2853 at T = 5 °C and T = 25 °C, revealing an increase in the protein content or a decrease in the lipid content, or both seeds above and below the optimum germination temperature and a statistically insignificant increase (large effect size (d = 0.80)) of unsaturation index (UI) at 5 °C and 25 °C, suggesting an increase of unsaturated lipids. The decrease in the ratio 1650/1540 at 5 °C (statistically significant) and 25 °C (statistically non-significant but with a large effect size (d = 0.80)) suggests changes in protein structure [58,59].

FTIR results also show a decrease in the ratio 1650/2853 in pH = 2 and pH = 12.5, suggesting a decrease in the protein content or an increase in the lipid content, or both seeds in ultra-acidic and very strongly alkaline environments [60], and a statistically insignificant decrease (large effect size (d = 0.80)) of UI, indicating a decrease of unsaturated lipids content in these conditions. The observed changes in seed metabolism could promote numerous signaling pathways that may be one of the principal causes of seedling inhibition, which is in line with the findings of Gill and Singh [61].

5. Conclusions

The current study was designed to explore the effect of abiotic factors (temperature, NaCl concentrations, and pH) on A. alpinus subsp. Meridionalis (Satureja) seed germination and metabolism. The optimum germination rate was found to be at T = 15 °C, pH = 7, and at low concentrations of NaCl. Furthermore, the molecular composition of germinated seeds subjected to extreme stresses was significantly different from the control. By revealing the optimal conditions and the changes occurring in seed metabolism, the findings of this study could be used to establish species-specific cultivation techniques and ex situ conservation strategies for this medicinal plant and contribute to the body of knowledge in some physiological events involved in the inhibition of the seed germination which is poorly understood and studies are scarce.