Identification of the NaCl-responsive metabolites in Citrus roots: A lipidomic and volatomic signature

ABSTRACT

It is known that the first osmotic phase affects the growth rates of roots immediately upon addition of salt; thus, dissecting metabolites profiling provides an opportunity to throw light into the basis of plant tolerance by searching for altered signatures that may be associated with tolerance at this organ. This study examined the influence of salt treatment on fatty acid composition and chemical composition of the essential oil of C. aurantium roots. Results proved that, under salt treatment, an increase of double bond index and linoleic desaturation ratio was pointed out. On the other hand, the reduction of saturated fatty acids was spotted. Such treatment also induced quantitative changes in the chemical composition of the essential oils from C. aurantium roots and increased markedly the rates of monoterpenes, while the sesquiterpenes decreased significantly. Both primary and secondary metabolites were found to be significantly salt responsive, including one fatty acid (palmitoleic acid) and six volatiles (E-2-dodecenal, tetradecanal, γ-Elemene, trans-caryophyllene, α-Terpinene and germacrene D). Plasticity at the metabolic level may allow Citrus plants to acclimatize their metabolic ranges in response to changing environmental conditions.

1. Introduction

Soil salinity is one of the most important abiotic stresses limiting the growth and crop production worldwide.¹ Recently, soil salinization reduced the world’s production of major crops by more than 50%. Thus, understanding salt-tolerance mechanisms and developing salt-tolerant crops are essential for maintaining the world’s food security.²

As a response to salt stress, plants suffer from osmotic stress and ion toxicity.³ The first “osmotic phase” affects the growth rates of shoots and roots immediately upon the addition of salt.^4,5

Although many studies exist on the effects of salinity on crop growth, few have investigated root-metabolic profiles,^6,7 and an even smaller number have conducted volatomics and/or lipidomics analyses of salt-stressed root tissues.^8,9

The majority of studies on the effects of abiotic stress on trees have focused on aboveground tissues and have neglected the impacts on belowground tissues.^10,11 This lack of information on roots, and root exudates, in particular, is largely due to their inaccessibility, so methods tend to be labor-intensive and have low precision (Brunner et al., 2015).

Being in direct contact with the soil solution, the roots are the first to encounter the saline medium.¹² The root distribution pattern in soil is the reflection of the plant ecological adaptation and may increase the chance of plant survival under stress.¹³

Plant exudates under abiotic stress consisted mainly of secondary metabolites (71% of total metabolites) associated with plant responses to stress, whereas the metabolite composition under recovery shifted toward a dominance of primary metabolites (81% of total metabolites). These results strongly suggested that roots exude the most abundant root metabolites.¹⁴ Moreover, root exudates comprise a large variety of compounds released by plants into the rhizosphere, including low-molecular-weight primary and secondary metabolites. Moreover, changes in exudate composition could have impacts on the plant itself, on other plants, on soil properties, and on soil organisms.¹⁴ Thus, dissecting metabolites profiling provides an opportunity to throw light into the basis of plant tolerance by searching for altered signatures that may be associated with tolerance. However, root responses to soil salinity in citrus species and their relation with plant growth are poorly understood.

Citrus, one of the most important fruit crops in the world, is sensitive to many environmental stresses including salt stress. Rootstocks greatly influence variety behavior as it ensures tolerance to abiotic stress conditions, as well as the provision of minerals and water for the total plant, and consequently impact crop yield and fruit quality. Rootstock choice is one of the most important decisions a grower makes when establishing commercial citrus orchards. The negative effects of stresses often lead to poor tree growth and reductions in fruit yield and quality.¹⁵ Thus, improvement of salinity tolerance, especially for rootstocks, can reduce economic loss to citrus growers. In spite of its economic importance, the metabolic mechanism of its extreme tolerance to salt stress is still a puzzle. So, a better understanding of the metabolic basis of the tolerance adaptation and resistance to high stressed environment is desired.

Accumulation of metabolites during abiotic stress is an important mechanism that is present in all the living organisms from microbes (e.g., bacteria and fungi) to higher plants and animals.¹⁶ The metabolic profiling has been reported in citrus plants in response to salt stress.^17,18

Understanding the physiological and metabolic mechanisms that confer salt tolerance of citrus rootstock is of agronomic and economic interest. The discovery of tissue tolerance traits could be used to select more salt‐tolerant varieties that maintain high yield under salt stress, and any positive traits could be transferred to other commercially important plants. In this study, the metabolite profiling analysis in the roots of C. aurantium after the exposure to salt stress has been performed using GC-MS analysis. Multivariate statistical tools have been employed to reduce and visualize the complex metabolomic datasets and to select the best salt-responsive metabolites.

2. Material and methods

2.1. Plant material and growth conditions

True-to-type C. aurantium rootstock plants were purchased from an authorized commercial nursery. One-year-old seedlings of the citrus plants were individually placed in plastic pots filled with perlite and watered three times a week with 0.5 L of a half-strength Hoagland solution. Plants were grown in the greenhouse under natural photoperiod and day and night temperature averaging 25.0 ± 3.0°C and 18.0 ± 3.0◦C, respectively, for 1 month to allow acclimation.

2.2. Stress treatments and experimental designs

After 1 month of acclimation in a greenhouse, plants were assigned at random into two blocks. According to availability, three to six plants were assigned for salt treatment and three plants were assigned as control plants. Salt stress was applied from July to the beginning of August 2017 under natural photoperiod conditions, with night/day temperatures ranging from 18°C to 38°C, and a relative humidity between 50% and 70%. Salt-treated plants were watered three times a week with a nutrient solution supplemented with 100 mM NaCl. To ensure that the treatments were uniformly applied, treatment was done at the same hour at field capacity. Harvesting was performed during day time and root parts were immediately submerged in liquid N2 to deter all the metabolic activity. The frozen roots were crushed and ground to fine powder and stored at −80°C for subsequent metabolomics analyses.

2.3. GC–MS for lipid profiling

Lipid extraction and determination were performed according to ISO method 6492 (1999). Methylation of FA followed the method of¹⁹. FA methyl esters were analyzed by an Agilent series 6890 capillary gas chromatography (Palo Alto, CA, USA). The separation was achieved on an HP-FFAP GC column (30 m length × 0.25 mm i.d. × 0.25 μm film thickness). Gas pressure was set at 1.2 bars for H₂, 1.5 bar for air and 1.0 bar for N₂, respectively. Column temperature was held at 80°C for 1 min after sample injection, increased to 200°C with a gradient of 12°C/min and held for 1 min, and further increased to 250°C with a gradient of 8°C/min and held for 5 min. The sample injection was performed in a split mode (split ratio: 50:1). The temperature of the detector and injector was 250°C and 230°C, respectively. The FA methyl esters were identified by comparing the retention times of the analytes with those of the standard substances and the relative contents were determined by the normalization of the chromatographic peak areas.

The double bond index (DBI) was calculated as follows: (1 x % monoenoic acids) + (2 x % dienoic acids) + (3 x % trienoic acids).²⁰

To evaluate the efficiency of the desaturation pathway during salt treatment,^21,22 the desaturation ratios from oleic to linoleic (ODR: oleic desaturation ratio) and from linoleic to linolenic acid (LDR: linoleic desaturation ratio) were calculated as follows:

ODR=[(%C18:2+%C18:3)/(%C18:1+C18:2+%C18:3)] x 100

LDR = [%C18:3/(%C18:2+%C18:3)] x 100

The magnitude of desaturation ratios represents the amount of substrate which is successfully desaturated from C18:1 to C18:2 and C18:3, thus providing a proportional measure of the desaturating enzymes’ activities during salt treatment.²¹

2.4. Extraction of volatile compounds and data processing

Samples (≈500 g) of citrus roots were suspended in water and subjected to hydrodistillation for 3 h using a Clevenger-type apparatus in accordance with European Pharmacopoeia method.²³ The steam pressure was fixed at three bars. When the boiling water begun and we got the first drop of EO, we regulate the temperature to a point where there was controlled boiling. The extracted essential oil was dried over anhydrous sodium sulfate (Na2SO4) and then stored at 4°C in brown glass vials. As a very sensitive, simple, and fast technique, gas chromatography–mass spectrometry (GC-MS) was selected for the analysis. Volatile compounds profiling was performed on a GC HP 5890 (II) interfaced with a Hewlett-Packard (HP) 5972 mass spectrometer with electron impact ionization (70 eV). An HP-5 MS capillary column (30 m × 0.25 mm, 0.25 mm film thickness; HP) was used using helium as a carrier gas at a flow rate of 1 mL/min. The program used was isothermal at 70°C, followed by 50–240°C at a rate of 5°C min1, then held at 240°C for 10 min. The split ratio was 60:1. Scan time and mass range were 1 s and 40–300 m/z, respectively.

Data processing and compound identification were performed as described in Ref.²⁴ Before the alignment step between chromatograms using the MetAlign software,²⁵ the raw data were treated by ChromaTOF software 2.0. The mass spectra of the representative masses were used for tentative identification by matching to the spectral NIST08 libraries and by comparison of the retention index calculated using a series of alkanes. Authentic reference standards were used to confirm the identity of the metabolites. Percentage compositions of samples were calculated according to the area of the chromatographic peaks using the total ion current.

2.5. Statistical analyses

Significant differences of metabolites between treatment and control were tested using T-test and ANOVA analysis on SPSS 21.0 software package. Treatment means within each measured parameter were separated by Duncan’s multiple range tests performed at a significance level of P ≤ 0.05. Quantitative normalization within replicates was transformed by logarithmic base of 2 and Metaboanalyst online analysis software (www.metaboanalyst.ca/) was used. Model parameters (goodness-of-fit R2Y and goodness-of-prediction Q2Y) were also produced. Regarding Q2Y prediction ability, a value >0.5 was adopted as a threshold to identify acceptable models, according to software recommendation and as set out in the literature.²⁶ Variable importance in projection (VIP analysis) was used to evaluate the importance of metabolites and to select those having the highest discrimination potential (VIP score >1). To achieve information on the regulation of biochemical processes related to salt treatment either under salinity or nonsaline control, a following fold-change analysis was performed for those metabolites highlighted by VIP analysis.

3. Results

Determination of the significantly altered metabolites accumulated upon salt stress was achieved with a targeted metabolite profiling analysis in Citrus species using GC-MS technique.

3.1. Lipidomic profile analysis upon control and salt stress treatments

Generally, after four weeks under stressful condition, the total lipid content increased in roots (Table 1). Total fatty acid (FA) content of Citrus roots was comprised of a range of FAs (C16 to C22); including 52.1% unsaturated and 42.9% saturated fatty acids (Table 1; Figure 1). Qualitatively spoken, the fatty acid profiles were similar; however, from a quantitative point of view, they displayed great quantitative differences (Table 1). At control condition (0 mM), FA composition was dominated with palmitic acid (C16:0; 27.55%) followed by linoleic acid (C18:2; 24.92%), oleic acid (C18:1; 12.24%), docosanoic acid (C22:0; 7.84%) and stearic acid (C18:0; 6.56%). The major changes in fatty composition in root parts were marked by the increase of the content of linoleic acid (C18:2), palmitoleic (C16:1) and erucic (C20:1) acids. However, an opposite trend of palmitic (C16:0) oleic (C18:1), docosanoic (C22:0), stearic acid (C18:0), arachidic (C20:0) and myristic (C14:0) acids was observed in Citrus roots. In general, NaCl treatment resulted in a remarkable increase of unsaturated fatty acids in root parts (Figure 1). In fact, the increase in lipid desaturation raised the DBIin root lipids. The same pattern was also evidenced by the increase of ODR in the root of salt-treated plants which reflects the higher efficiency of the desaturation system from linoleic to linolenic acid. The results suggested that unsaturated fatty acids, particularly palmitoleic (C16:1), erucic (C20:1) and oleic (C18:1) acids, might play a role in the defense mechanism against salinity stress during plant development. Specifically, Citrus plants respond to biotic stress by remodeling membrane fluidity and by releasing palmitoleic acid (C16:1) from root membrane lipids.

Table 1.

Changes in fatty acids (FA) composition, expressed as total fatty acids (%), in C. aurantium roots treated during 1 month with different 100 mM NaCl.

Fattyacids	%		Fold changes log2(salt/control)
	0 mM NaCl	100 mM NaCl
Palmiticacid (C16:0)	27.55 ± 1.25	24.33 ± 1.11	−0.18
Docosanoicacid (C22:0)	7.84 ± 0.02	5.02 ± 0.05	−0.64
Stearicacid (C18:0)	6.56 ± 0.25	5.8 ± 0.21	−0.18
Arachidicacid (C20:0)	4.74 ± 0.15	2.96 ± 0.05	−0.68
Myristicacid (C14:0)	1.48 ± 0.05	0.81 ± 0.02	−0.87
Pentadecanoic Acid (C15:0)	0	1 ± 0.05	_
PalmitoleicacidC16:1)	3.24 ± 0.02	32.44 ± 1.32	3.32
Erucicacid (C20:1)	1.18 ± 0.05	1.22 ± 0.02	0.05
Oleicacid (C18:1)	12.24 ± 0.05	6.66 ± 0.15	−0.88
Linoleicacid (C18:2)	24.92 ± 1.05	31.87 ± 1.23	0.35
Methy l linolenate (C18:3)	1.42 ± 0.03	1.21 ± 0.05	−0.23
SFA	48.17	39.92
UFA	43.00	73.4
DBI	70.76	107.69
ODR	68.27	83.24
LDR	5.39	3.66

SFA, saturated fatty acids; UFA, unsaturated fatty acids; DBI, double bond index, ODR, oleic desaturation ratio; LDR, linoleic desaturation ratio.

Values are means ± SD of triplicate.

Figure 1.

Changes of fatty acids (FA) structural groups, DBI (double bond index), ODR (oleic desaturation ratio) and LDR (linoleic desaturation ratio) in C. aurantium roots under salt stress.

3.2. Volatomic profile analysis upon control and salt stress treatments

Table 2 shows the identity, retention index and percent composition of the identified volatiles obtained from the roots of treated and non-treated Citrus plants. Comparison of the analytical data of the oils revealed significant qualitative and quantitative differences in the composition of the root tissues. Sixteen different compounds were identified in the roots of control plants. The most of which were aldehydes (35.49%) comprising E-2-dodecenal (30.81%), and terpenic alcohols and aldehydes (19.88%) represented mainly by thymol (12.6%). Under salt stress, remarkable quantitative changes in the chemical composition of the essential issued from Citrus roots were observed. In fact, the proportions of 8 metabolites were over accumulated under 100 mM NaCl, while 2 were decreased. Salinity stress at 100 mM enhanced the rates of aldehydes, monoterpene biosynthesis. It is very interesting to note that among aldehydes, the biosynthesis of E-2-dodecenal reached a maximum level (37.60%). On the other hand, under the same treatment, a decrease in the percentages of terpenic alcohols and aldehydes and sesquiterpenes was noted (Figure 2). At 100 mM NaCl treatment, the biosynthesis of new compounds like α-terpinene, germacrene D, linalyl acetate, terpinen-4-ol and trans-p-Menth-2-en-1-ol was induced. On the other hand, six of the identified metabolites (γ-Elemene, trans-caryophyllene, (E)-2-decenal, β-Caryophyllene, geraniol and myrcene) were totally suppressed under 100 mM salt-treated plants. These latter were mainly sesquiterpenes (γ-Elemene, trans-caryophyllene and β-Caryophyllene), two compounds from the terpenic alcohols and aldehyde class (thymol and geraniol), an aldehyde represented by (E)-2-decenal and a monoterpene (myrcene) (Table 2). The highest fold changes (>2) were detected for Linalool (3.41) and for terpinolene (2.81).

Table 2.

Effects of salt treatment on volatile composition (% peak area) of C. aurantium roots.

RIa	RIb	Compounds	% Peak area		Fold changes log2(salt/control)
			0 mM NaCl	100 mM NaCl
939	1032	α-Pinene	0.81 ± 0.05	0.49 ± 0.02	−0.73
1018	1186	α-Terpinene	0	4.2 ± 0.11	_
1088	1186	Terpinolene	0.07 ± 0.01	0.49 ± 0.05	2.81
994	1174	Myrcene	0.08 ± 0.01	0
1143	1532	Camphor	0.24 ± 0.06	0.76 ± 0.01	1.66
1189	1693	α-Terpineol	0.42 ± 0.03	0.17 ± 0.05	−1.30
1178	1611	Terpinen-4-ol	0	0.21 ± 0.02	_
1281	1792	(E)-2-decenol	0.51 ± 0.09	0.78 ± 0.12	0.61
1466	1792	Dodecen-11-1-ol	5.98 ± 0.21	7.16 ± 0.21	0.26
1098	1553	Linalool	0.05 ± 0.02	0.53 ± 0.02	3.41
1255	1857	Geraniol	0.11 ± 0.05	0	_
1142	1571	Trans-p-Menth-2-en-1-ol	0	0.19 ± 0.01	_
1262	1855	E-2-dodecenal	30.81 ± 1.25	37.6 ± 1.15	0.29
1265	1653	(E)-2-decenal	0.5 ± 0.05	0	_
1620	1919	Tetradecanal	2.85 ± 0.35	6.78 ± 0.52	1.25
1310	1605	Undecanal	1.33 ± 0.02	1.91 ± 0.05	0.52
1339	1650	γ-Elemene	4.2 ± 0.05	0	_
_	1414	Trans caryophyllene	4.2 ± 0.05	0	_
1418	1612	β-Caryophyllene	0.49 ± 0.035	0	_
1480	1727	Germacrene D	0	3.94 ± 0.07	_
1262	1556	Linalyl acetate	0	0.26 ± 0.02	_
		Monoterpenes	1.2	5.94
		Terpenic alcohols	7.07	9.04
		Aldehydes	35.49	46.29
		Sesquiterpenes	8.89	3.94
		Esters	0	0.26

Values are means ± SD of triplicate.

RI retention indices relative to n-alkanes on ^aHP-5 and ^bHP-Innowax columns.

Figure 2.

Alterations of the percentages of the main representative classes of the essential oil isolated from C. aurantium roots under salt stress.

3.3. Identification of the salt-responsive metabolites

Using a powerful pattern recognition method, a supervised PLS-DA (Partial Least Square Discriminant Analysis) was performed. The prediction accuracies were assessed by cross-validation with different numbers of components. Taking into account the lipidomic profile, satisfactory modeling and prediction results were already gained with two PCs (accuracy 1, R² > 0.90, Q² > 0.9) when data were analyzed using control and salt-stressed samples. This indicates that metabolomes under control and salt-stress conditions are largely distinguishable in Citrus roots. When based on volatomic profile, the same trend of group separation was observed for control vs. stressed Citrus root comparison (accuracy 1, R² > 0.90, Q² > 0.9), indicating a metabolic perturbation under salt stress in the studied samples. PLS-DA also, as a supervised method, allows the selection of the most predictive or discriminative features that are potentially useful in helping sample classification. Fundamentally, a measure of the variable importance in the PLS-DA is the VIP (variable of importance in prediction) score. Accordingly, VIPvalues greater than 1 were considered the most relevant metabolites for explaining the responses.²⁷ On the basis of the parameter VIP > 1, one fatty acid and six volatiles with significant changes (student’s T-test P < .05), were, respectively, identified as the most powerful responsive metabolites (Table 3). Among fatty acids, the changed metabolite was palmitoleic acid (C16:1) was selected as the best responsive metabolite. These latter were over-accumulated under salt-stress treatment. Compared to the non-treated control, two metabolites were over accumulated in Citrus roots after salt exposure; two volatiles were newly synthesized, while two were totally inhibited. Compared to the non-treated control, the identified volatile-responsive metabolites are divided into over-accumulated (E-2-dodecenal andtetradecanal), inhibited (γ-Elemene and trans-caryophyllene) and newly synthesized volatiles (α-Terpinene and germacrene D) (Table 3).

Table 3.

List of the compounds selected as the best salt-responsive metabolites for C. aurantium roots.

Name	VIP value	f value	p value
Palmitoleicacid	3.1155	3.65E+31	2.73E-123
E-2-dodecenal	2.7044	4.91E+30	2.14E-121
α-Terpinene	1.6629	7.50E+30	7.00E-125
γ-Elemene	1.6629	1.46E+31	1.92E-123
Trans caryophyllene	1.6629	1.22E+31	3.93E-122
Tetradecanal	1.5653	1.26E+31	7.57E-121
Germacrene D	1.5593	3.58E+30	5.70E-123

Higher VIP values indicate a stronger influence of the metabolite in distinguishing different groups.

4. Discussion

Under salt-induced stress, plants are primarily affected by disturbances in the osmotic potential of the rhizosphere, which is caused by higher salt levels.²⁸ Besides, the root architecture is important for plants to access soil resources, and morphological and physiological adaptation of the root system under stress conditions may result in the continuation of nutrient absorption and utilization.²⁹ Most studies on root uptake have assumed that the entire root is equally active.³⁰ It is recognized that the deleterious effects of salt stress were not limited to some physiological traits, but they can affect other biochemical processes namely primary and secondary metabolisms.^31,32

Mass spectrometry–based lipid profiling has been used to track the changes in lipid levels and related metabolites in response to salinity stress.³² The analytical data revealed that fatty acid profiles were qualitatively similar between control and salt-treated plants, but they displayed great quantitative differences. Further, under salt conditions, a redirection of the lipidic metabolism toward synthesis of unsaturated fatty acids was evidenced by the increase of DBI and ODRsuggesting hence, that under salt conditions, Citrusplant may allocate its unsaturated fatty acids to the root parts. Such root-metabolic feature could be associated with the need of maintaining a high degree of unsaturation in the root tissues. In fact, known as a main component of plasma membrane lipids, fatty acids are considered to be important in salt tolerance of plants, and a high level of membrane lipid unsaturation is required to maintain the membrane fluidity necessary for proper membrane functions.³³ The obtained results are in agreement with previous works that showed an increased level of unsaturation index in response to salt stress for other species like broccoli³⁴ and safflower.³¹ The response to salt damage may be regulated by an increased content of C18:2 and degree of unsaturation.^31,35 Other studies showed that salt-resistant maize and barley subjected to salt stress commonly had increased levels of C18:3 and unsaturation degree in their membranes compared with salt-sensitive ones.^32,36 These results are in accordance with previous studies that show that the ability to maintain or increase unsaturated lipids correlates with a high level of salinity tolerance.^34,37 The ability of wheat and several algae to tolerate salinity has also been shown to be due to increased unsaturated fatty acid.^38,39 There are certainly differences in changes in the fatty acid composition among species growing in the saline environment, but increased unsaturation degree of membrane is the key to species salt tolerance. Therefore, we can regard it as a common salt-responsive strategy adopted in C. aurantium rootstock. This work demonstrated that response to salt stress depends on plant species and that from a practical viewpoint; it appears that salt treatment could be considered as a promising alternative to obtain good quality oil in terms of abundance of unsaturated fatty acids in Citrus species. There is almost no information or reports concerning the composition of Citrus roots essential oils and about the NaCl effects on these compositions. In the present study, the main changes caused by NaCl treatments were related to the relative proportion of these secondary metabolites, synthesis of new ones and the disappearance of others. Regarding the chemical composition of the essential oil, our results showed that salt treatment enhanced the production of terpinolene (2.81-fold), camphor (1.66-fold), linalool (3.41-fold) in Citrus roots. Meanwhile, α-Pinene (−0.73-fold) and α-Terpineol (−1.30-fold) were significantly decreased. It is notable that in Citrusroots, the increased amounts of oxygenated components represented the main variations in volatile oil constituents suggesting the enhancement of the activities of related biosynthetic enzymes.^40,41 Thus, such compositional changes in essential oil composition could reflect an adaptive mechanism to cope with the deleterious effects of salinity. Oxygenated compounds are known for their potent biological activities⁴² and their high capacity to scavenge the active oxygen species (AOS);⁴³ hence, the over-accumulation of the above-mentioned metabolites, namely terpinolene and linalool in roots may have a defensive role against AOS induced by salt treatment.⁴⁴

5. Conclusion

Our findings suggest that high-throughput GC-MS methods combined with chemometrics are suitable for seeking the “key stress-associated metabolites” associated with environmental change and understanding the adaptation mechanism against extreme environmental stress. This study demonstrated that metabolomics could provide an insightful view of the small-molecule differential metabolites of Citrus undersalt stress. It assists us in understanding adaptation under extreme environmental stress and will ultimately benefit future breeding programs for salt-tolerant genotypes. Conclusions drawn here revealed that C. aurantiumrootstockexhibited specific mechanisms for salinity tolerance based on the alteration in the fatty acid and volatile profile composition. In fact, it was demonstrated that salinity could induce C. aurantium roots to produce high amounts of some valuable fatty acids and volatile oils. Therefore, the development of new chemotypes at different salt levels could be considered as valuable aspects of salinity stress in some plants inducing them to produce compounds with industrial and pharmaceutical interest.

Funding Statement

This work was financially supported by the Tunisian Ministry of Higher Education and Scientific Research.

Conflicts of interest

There are no conflicts of interest to declare.