Omeprazole Promotes Chloride Exclusion and Induces Salt Tolerance in Greenhouse Basil

Abstract

The role of small bioactive molecules (<500 Da) in mechanisms improving resource use efficiency in plants under stress conditions draws increasing interest. One such molecule is omeprazole (OMP), a benzimidazole derivative and inhibitor of animal proton pumps shown to improve nitrate uptake and exclusion of toxic ions, especially of chloride from the cytosol of salt-stressed leaves. Currently, OMP was applied as substrate drench at two rates (0 or 10 μM) on hydroponic basil (Ocimum basilicum L. cv. Genovese) grown under decreasing NO₃⁻:Cl⁻ ratio (80:20, 60:40, 40:60, or 20:80). Chloride concentration and stomatal resistance increased while transpiration, net CO₂ assimilation rate and beneficial ions (NO₃⁻, PO₄³⁻, and SO₄²⁻) decreased with reduced NO₃⁻:Cl⁻ ratio under the 0 μM OMP treatment. The negative effects of chloride were not only mitigated by the 10 μM OMP application in all treatments, with the exception of 20:80 NO₃⁻:Cl⁻, but plant growth at 80:20, 60:40, and 40:60 NO₃⁻:Cl⁻ ratios receiving OMP application showed maximum fresh yield (+13%, 24%, and 22%, respectively), shoot (+10%, 25%, and 21%, respectively) and root (+32%, 76%, and 75%, respectively) biomass compared to the corresponding untreated treatments. OMP was not directly involved in ion homeostasis and compartmentalization of vacuolar or apoplastic chloride. However, it was active in limiting chloride loading into the shoot, as manifested by the lower chloride concentration in the 80:20, 60:40, and 40:60 NO₃⁻:Cl⁻ treatments compared to the respective controls (−41%, −37%, and −24%), favoring instead that of nitrate and potassium while also boosting photosynthetic activity. Despite its unequivocally beneficial effect on plants, the large-scale application of OMP is currently limited by the molecule’s high cost. However, further studies are warranted to unravel the molecular mechanisms of OMP-induced reduction of chloride loading to shoot and improved salt tolerance.

1. Introduction

Soil salinity affects plants’ growth and yield via pleiotropic mechanisms involving water stress [1,2,3], ion toxicity and nutrient imbalance [4,5,6,7], and oxidative damage [8,9]. When the electrical conductivity (EC) of the soil exceeds the threshold limit of 4 dS m⁻¹, corresponding to about 40 mM of sodium chloride (NaCl), most of the glycophytes, among which vegetable crops, undergo a reduced capacity to absorb water from soil [10,11]. This latter effect translates into a disturbance in plant water relations, stomatal aperture and transpiration that reduces the expansion of existing leaves and the emergence of new leaves and lateral buds [12]. If the stress persists, excess ions accumulate in the plants causing osmotic and ion specific effects, which interfere with protein synthesis and activity, membrane stability, and electron transport thereby impairing growth and crop productivity [2,12]. One ubiquitous protective mechanism involves the synthesis of small molecules (<500 Da) that are directly or indirectly involved in osmotic balance, and stabilization and protection of membranes and macromolecules by oxidative stress [3,13,14]. Most of these small molecules are nitrogen-containing compounds, such as amino acids, amines, and betaines, or sugars, which, when accumulated in the cytosol, counteract the osmotic and toxic effect of sodium and chloride mostly compartmentalized in the vacuole [15,16,17].

Moreover, biologically active small molecules, of natural or synthetic origin, can be supplied exogenously at very low concentrations and relieve or protect plants from saline stress [18,19,20,21]. Several recent studies have highlighted a beneficial role of omeprazole (OMP), a small molecule of 345 Da, in salinity tolerance [22,23,24,25] and mechanical stress tolerance [26]. OMP is a benzimidazole derivative used as a prescribed drug to treat peptic ulcers, gastroesophageal reflux, and other hypersecretory states in humans for its ability in inhibiting the P-type IIC proton pump H⁺/K⁺-ATPases in the gastric parietal cell, blocking the final step in the gastric acid secretory pathway [27,28,29]. Notwithstanding, there is a very low homology between plants and animal proton pumps [22], it has been shown that OMP can act as plant growth regulator improving tolerance to salinity at concentration ranging between 10 and 100 μM [22,23]. OMP does not directly regulate potassium to sodium ratio and ion homeostasis, but it is able to increase the concentration of nitrate in roots and leaves while reducing those of sodium and chloride in both organs. This increase of nitrate can favor the synthesis and accumulation of N-containing compounds involved in osmotic adjustment and ROS scavenging, improving the physiological functions and viability of salt stressed plants [3]. Moreover, OMP is able to increase root growth and nutrients resource efficiency in control plants [3] and mechanical stressed plants [26]. The involvement of OMP in the regulation of plant radical growth and protection from salt stress certainly entails a complex signaling and/or hormonal action [3,23], determined at the genetic level, as demonstrated by the recent work of Cirillo et al. [25] on two basil contrasting genotypes, that, though, has not yet been unraveled. Cirillo et al. [25] attributed the OMP beneficial effect on the salt sensitive basil genotype Napoletano to this small molecule specific capacity to improve chloride exclusion from the cytosol of salt stressed plants. However, their study, as well as the most of the studies performed on the effects of salinity on plants, have used NaCl as salt, and focused on sodium accumulation and toxicity, considering sodium as the main problem responsible for the reduction of plant growth, development, and survival [5,30,31,32,33,34,35]. Indeed, sodium can reduce potassium and calcium uptake, affecting stomatal conductance; it can substitute potassium in key enzymatic reactions leading to enzyme inhibition, alteration of metabolic processes, plant nutritional imbalance and oxidative stress [23,36,37]. However, chloride can be toxic as well when accumulated in the cell at high concentrations. Nonetheless, there is a paucity of literature that documents the specific molecular and physiological mechanisms that lead to chloride adverse effects on plants [38,39].

Chloride is a micronutrient that in non-saline conditions is beneficial for plants; in fact, it accumulates to macronutrient levels through a secondary active transport, an electrogenic Cl^–/2H⁺ symport mechanism ([40] and references therein). When present in a range of 0.2–2 mg g⁻¹ fresh weight (fw) [41,42], in addition to the well-known functions in the stabilization of the oxygen evolving complex of photosystem II (PSII), it has a key role in the maintenance of cell membranes electric potential, regulation of tonoplast H⁺-ATPase, pH-stat, amylases, and asparagine synthetase ([43] and references therein). Chloride has also been found to have adaptive functions improving water homeostasis in tobacco at concentration up to 4 mg g⁻¹ fw [44]. However, higher chloride concentration can be toxic impairing PSII quantum yield and photosynthetic electron transport rate and consequently causing other physiological dysfunctions in plants [45,46].

Taking also into account that a practice adopted in horticulture is the decrease of calcium nitrate in the culture media and its substitution with calcium chloride to meet the EU directives aimed at reducing the accumulation of nitrate in leafy vegetables and its possible implications to human health [47]. Thus, it is of pivotal importance to understand which morphological and physiological processes are specifically impaired by chloride in excess and decrease of nitrate, and if and how the addition of OMP can alter ions/chloride homeostasis in plant tissues allowing plants to overcome the negative effects of chloride salinity. To verify this hypothesis, a greenhouse experiment was conducted to test the effect of decreasing NO₃⁻:Cl⁻ ratio in absence or presence of OMP on morpho-physiological and ion homeostasis responses of basil (Ocimum basilicum L.) grown in soilless culture.

2. Materials and Methods

2.1. Greenhouse Conditions, Plant Material, and Experimental Design

The trial was conducted during the growing season 2017, in a glasshouse situated at the experimental station of the Department of Agricultural Sciences, University of Naples Federico II located at Torre Lama (Bellizzi; 43°31′ N, 14°58′ E; 60 m above sea level). The tested crop for the present experiment was basil (Ocimum basilicum L., family Lamiaceae) cv. Gecom (Genovese type; Società Agricola Italiana Sementi-SAIS, Cesena, Italy). Basil plants were grown hydroponically in an open-loop soilless system using a mixture of peat and perlite as substrate. Basil seedlings were transplanted on May 9, into plastic pots containing 1.3 L of a mixture peat and perlite in a 2:1 (v:v) ratio. Plastic pots were disposed on aluminum benches (1.8 m × 8.0 m) in single row at plant density of 22 plants per square meter (15 × 30 cm). Inside the glasshouse, the daily mean temperature was maintained between 16 and 27 °C and the relative humidity ranged between 55% and 80% during the growing cycle.

Lowering the NO₃⁻:Cl⁻ ratio of the nutrient solution is a common practice in horticulture used to reduce nitrate content and improve leafy vegetables quality without altering the ionic strength of the culture media. However decreasing NO₃⁻:Cl⁻ ratio can affect the morpho-physiological and qualitative features of salt-sensitive crops. Therefore, the glasshouse experiment was designed as a factorial combination of four ratios of nitrate (NO₃⁻) to chloride (Cl⁻) (80:20, 60:40, 40:60, or 20:80) and two omeprazole (OMP) application levels (0 or 10 µM) to verify if OMP could ameliorate the status of plants even at very low NO₃⁻:Cl⁻ ratio. The eight treatments were arranged in a complete randomized block design, with three replicates per treatment. Each experimental unit (replication) consisted of 10 basil plants.

2.2. Nutrient Solution Management, NO₃⁻: Cl⁻ Treatments, and Omperazole Application

The concentrations of macro (in mM) and micronutrients (in µM) in the nutrient solution were: 1.5 mM P, 4.5 mM K, 6.5 mM Ca, 2 mM Mg, 20 µM Fe, 9 µM Mn, 0.3 µM Cu, 1.6 µM Zn, 20 µM B, and 0.3 µM Mo [47]. The four NO₃⁻:Cl⁻ treatments studied in the current experiment were obtained by adding different salt quantity of calcium nitrate (Ca[NO₃]₂) and calcium chloride (CaCl₂) to the above nutrient solution. Particularly, the nitrate (NO₃⁻), Ca(NO₃)₂, and CaCl₂ salt quantity in the nutrient solution were (i) 9.6 mM, 867.1 and 133.2 mg L⁻¹ for 80:20 treatment, (ii) 7.2 mM, 650.3 and 266.4 mg L⁻¹ for 60:40 treatment, (iii) 4.8 mM, 433.5 and 399.6 mg L⁻¹ for 40:60 treatment and (iv) 2.4 mM, 216.8 and 532.8 mg L⁻¹ for 20:80 treatment. The electrical conductivity (EC) and the pH of the nutrient solution in all treatments were 1.9 ± 0.2 dS m⁻¹ and 6.0 ± 0.2, respectively. The nutrient solution in the four NO₃⁻:Cl⁻ treatments were pumped from independent 50 L plastic tanks and delivered to the plants through a drip irrigation system with one auto-compensating dripper per plant having a flow rate of 2 L h⁻¹.

The OMP was supplied as root application five times during the cropping season starting on May 19 (11 days after transplanting, DAT) at seven-days interval (26 May, 2, 9, and 16 June). The OMP application was delivered manually at a uniform rate level of 100 mL per pot.

2.3. Biometric and Leaf Color Measurements

Forty-four DAT, the number of leaves per plant was recorded on six basil plants per experimental unit. Total leaf area was measured by an electronic area meter (LI-COR 3100C biosciences, Lincoln, NE, USA). Then, plants were divided in leaves, stems and roots, where the fresh yield was determined. Samples of fresh leaf, stem and root were dried at 70 °C for 72 h until they reached a constant weight, for shoot (leaves + stems) and root dry biomass assessment. The root-to-shoot ratio as well as the leaf dry matter content (%) were also calculated.

The leaf color components in particular brightness (L*), redness (a*) and yellowness (b*) were also measured on the upper leaf part of six basil plants per experimental unit using an 8 mm-aperture Minolta CR-300 Chroma Meter (Minolta Camera Co. Ltd., Osaka, Japan). The calibration of the Chroma Meter was carried out just before leaf color sampling as described in detail by Colonna et al. (2016).

2.4. Soil Plant Analysis Development Index and Leaf Gas Exchange

One day before the termination of the experiment (June 20; 43 DAT), the SPAD index was measured on twenty fully developed basil leaves per replicate as described in detail by Kumar et al. [48] using a portable chlorophyll meter SPAD-502 (Konica, Minolta, Tokyo, Japan).

On the same date, all the physiological parameters including net carbon dioxide assimilation rate, stomatal resistance, transpiration rate were measured with a portable gas exchange analyzer (LCA-4; ADC BioScientific Ltd., Hoddesdon, UK) equipped with a broadleaf chamber (cuvette window area of 6.25 cm²) [49]. Similarly, to SPAD index, the leaf gas exchange measurements were carried out on fully expanded basil leaves, using six replicates per experimental unit. Instantaneous water use efficiency was calculated after dividing net CO₂ assimilation rate by transpiration rate [3].

2.5. Ion Analysis of Leaf Tissue

Total nitrogen was determined by the established Kjeldahl method [50], whereas cations and anions measurements in leaf tissue were performed according to the procedure described by Rouphael and co-workers [49] and Kyriacou et al. [51]. Total N as well as cations and anions determination were carried out on dried leaf samples, which were ground with an electrical mill to an 841 µm mesh. Briefly, two hundred fifty grams of dried leaf tissue were suspended in 50 mL of ultrapure water (Milli-Q, Merck Millipore, Darmstadt, Germany), and incubated at 80 °C for 10 min in a shaking water bath (ShakeTemp SW22, Julabo, Seelbach, Germany). The suspensions were first centrifuged (at 6000 rpm for 10 min) then filtered before injection (25 µL) in the ion chromatography (ICS-3000, Dionex, Sunnyvale, CA, USA) coupled to a conductivity detector. The implemented columns used in the current experiment were: 1) IonPac CG12A (4 250 mm, Dionex, Sunnyvale, CA, USA) guard column and IonPac CS12A (4 × 250 mm, Dionex, Sunnyvale, CA, USA) analytical column for cations (K⁺, Ca²⁺, Mg²⁺ and Na⁺) determination, 2) IonPac AG11-HC (4 × 50 mm) guard column and IonPac AS11-HC (4 × 250 mm) analytical column for anions (NO₃⁻, PO₄³⁻, SO₄²⁻, and Cl⁻) analysis.

2.6. Statistics

The experimental data were subjected to a two-way analysis of variance (ANOVA) to determine the main effects (different NO₃⁻:Cl⁻ ratios and OMP application) as well as the interaction between these two tested factors. OMP application main effects were compared by t-Test. Duncan’s multiple range test was performed for mean comparisons on each of the significant (p < 0.05) variables measured. The software package SPSS 20 was used for the ANOVA. The Principal Component Analysis (PCA) was also conducted on morpho-physiological and mineral composition to determine the score plot and loading matrix based on the first and second principal components. The PCA analysis was assess using Minitab 16.2.1 statistical software [3,52]. The score plot and loading matrix were also determined based on the first and second principal components (PCs). A heatmap was generated using the https://biit.cs.ut.ee/clustvis/ online program package with Euclidean distance as the similarity measure and hierarchical clustering with complete linkage. Morpho-physiological parameters and mineral composition data were visualized using a false color scale, with red indicating an increase and blue a decrease of values [3,24]. A different heat map generated in Excel was used to summarize the plant responses to different nutrient compositions and OMP. Results were calculated as Log2 of 60:40, 40:60, and 20:80/80:20 in control plants, and Log1.3 of 10 µM OMP/0 µM OMP control in plants grown at different N. Results were visualized using a false color scale with red indicating an increase and blue a decrease, while no differences were visualized by white squares [53].

3. Results

3.1. Morphological Responses Induced by OMP under Different NO₃⁻:Cl⁻ Ratios

In this experiment, for fresh yield, shoot and root dry biomass significant interactions (p < 0.01) were recorded between the two tested factors (NO₃⁻:Cl⁻ ratio and omeprazole [OMP] application). Fresh yield, shoot and root dry biomass were higher in OMP-treated basil plants with a varied response in terms of NO₃⁻:Cl⁻ ratio in the nutrient solution (Figure 1). In particular, basil plants treated with 10 µM OMP and grown under both 80:20 and 60:40 NO₃⁻:Cl⁻ ratio presented the highest marketable yield, shoot, and root biomass (Figure 1). Interestingly, we observed contrasting responses in treated and untreated basil plants with increasing Cl⁻ concentration in the nutrient solution (e.g., by decreasing NO₃⁻:Cl⁻ ratio). For instance, decreasing NO₃⁻:Cl⁻ ratio from 80:20 to 20:80 showed a linear reduction in the tested morphometric traits compared to the control treatment (80:20). Whereas the responses to the decrease of NO₃⁻ and simultaneous increase of Cl⁻ in conjunction to OMP diverged greatly; since no significant reduction in fresh yield, shoot and root dry biomass was observed in OMP-treated plants grown under 60:40 NO₃⁻:Cl⁻ ratio compared to the control treatment. Moreover, even in plants grown at 40:60 NO₃⁻:Cl⁻ ratio, fresh yield did not decrease compared to the control without OMP (Figure 1).

Irrespective of OMP application (no significant interaction between the two tested factors), the number of leaves, total leaf area and root-to-shoot (R/S) ratio of basil declined under the concomitant NO₃⁻ decrease and Cl⁻ increase in the nutrient solution with the lowest values observed in 20:80 NO₃⁻:Cl⁻ ratio (e.g., severe chloride conditions) (

Table 1. Effects NO₃⁻:Cl⁻ ratio in the nutrient solution and omeprazole (OMP) application rates on growth parameters, root-to-shoot ratio and leaf colorimetry of greenhouse hydroponic basil plants.

Source of variance.	Leaf Number	Leaf Area	Root-to-Shoot Ratio	Leaf Dry Matter	L*	a*	b*
	(no. plant−1)	(cm2 plant−1)		(%)
NO3−:Cl− (N)
80:20	103 a	1130 a	0.11 a	8.73 c	41.08 c	−15.73 a	21.11 c
60:40	87 b	1196 a	0.11 a	8.78 c	42.34 c	−16.20 ab	21.20 c
40:60	84 b	983 b	0.09 b	9.56 b	44.40 b	−16.92 b	23.14 b
20:80	73 c	751 c	0.07 c	11.18 a	46.49 a	−18.19 c	25.87 a
	***	***	***	***	***	***	***
Omeprazole (μM) (OMP)
0	81	955	0.08	9.62	43.66	−17.13	24.03
10	93	1076	0.11	9.51	43.49	−16.39	21.62
t-test	0.030	0.150	0.001	0.832	0.876	0.125	0.021
N × OMP
80:20 × 0 μM OMP	98	1123	0.10	9.16	41.98	−16.42	22.04
60:40 × 0 μM OMP	78	1124	0.09	8.71	42.19	−16.55	21.64
40:60 × 0 μM OMP	77	895	0.08	9.66	44.22	−17.20	24.72
20:80 × 0 μM OMP	71	678	0.06	10.93	46.25	−18.35	27.74
80:20 × 10 μM OMP	109	1138	0.12	8.31	40.19	−15.03	20.18
60:40 × 10 μM OMP	95	1268	0.13	8.86	42.48	−15.85	20.76
40:60 × 10 μM OMP	91	1072	0.11	9.46	44.57	−16.63	21.55
20:80 × 10 μM OMP	75	825	0.08	11.42	46.73	−18.03	24.01
	ns	ns	ns	ns	ns	ns	ns

ns, *** not significant or significant at p ≤ 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple-range test (p = 0.05). Omeprazole application rates are compared according to Student’s t-test.

). In addition, when averaged over NO₃⁻:Cl⁻ ratio in the nutrient solution, the leaf number as well as the R/S ratio of OMP-treated basil plants were higher than those of untreated control by 14.8% and 37.5 %, respectively (Table 1).

Leaf yellow intensity expressed by positive and b* values, was significantly affected by NO₃⁻:Cl⁻ ratio and OMP treatments with no significant interaction between the two factors, whereas the leaf brightness (L*) and leaf green intensity (i.e., negative a* values) were only influenced by the ratio between NO₃⁻ and Cl⁻ when this latter was in strong favor to Cl⁻ in the nutrient solution (Table 1). Specifically, Cl⁻ concentration in the nutrient solution yielded lighter basil leaves, expressed by increasing L* values. Regardless, the NO₃⁻:Cl⁻ ratio, OMP-treated plants exhibit lower leaf yellow intensity values compared to untreated control (Table 1).

3.2. Physiological Responses Induced by OMP under Different NO₃⁻:Cl⁻ Ratios

SPAD index and leaf gas exchange as a function of NO₃⁻:Cl⁻ ratio and OMP application are reported in

Table 2. Effects NO₃⁻:Cl⁻ ratio in the nutrient solution and omeprazole (OMP) application rates on physiological parameters: SPAD index, net CO₂ assimilation (A_CO2), stomatal resistance (r_s), transpiration rate (E) and instantaneous water use efficiency (WUE_i) of greenhouse hydroponic basil plants.

Source of Variance	SPAD Index	ACO2	rs	E	WUEi
		(μmol CO2 m−2 s−1)	(m−2 s−1 mol−1)	(mol H2O m−2 s−1)	(μmol CO2 mol−1 H2O)
NO3−:Cl− (N)
80:20	39.59 a	11.89 a	5.56 b	3.81 a	3.16
60:40	38.35 ab	11.67 ab	8.01 a	3.29 ab	3.60
40:60	37.25 b	10.60 bc	7.52 ab	3.09 b	3.48
20:80	31.59 c	9.57 c	8.35 a	2.96 b	3.32
	***	**	*	*	ns
Omeprazole (μM) (OMP)
0	35.64	10.22	8.10	3.45	2.98
10	37.74	11.64	6.61	3.13	3.79
t-test	0.145	0.014	0.050	0.126	0.001
N × OMP
80:20 × 0 μM OMP	38.27	11.71	6.34	4.03	2.92
60:40 × 0 μM OMP	37.04	11.02	8.41	3.38	3.29
40:60 × 0 μM OMP	36.47	9.87	8.10	3.22	3.09
20:80 × 0 μM OMP	30.79	8.29	9.56	3.16	2.63
80:20 × 10 μM OMP	40.90	12.06	4.78	3.59	3.41
60:40 × 10 μM OMP	39.65	12.32	7.60	3.20	3.90
40:60 × 10 μM OMP	38.04	11.33	6.93	2.97	3.87
20:80 × 10 μM OMP	32.38	10.86	7.14	2.75	4.00
	ns	ns	ns	ns	ns

ns, *, **, *** not significant or significant at p ≤ 0.05, 0.01 or 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple-range test (p = 0.05). Omeprazole application rates are compared according to Student’s t-test.

. No significant interaction between the two tested factors were observed in the studied physiological parameters. When averaged over OMP application, increasing the Cl⁻ concentration in the nutrient solution reduced linearly the SPAD index and net CO₂ assimilation rate (A_CO2) as well as the transpiration rate (E), while it increased the stomatal resistance (r_s) (Table 2). Moreover, irrespective NO₃⁻:Cl⁻ ratio, substrate drench application of 10 µM OMP increased A_CO2 and instantaneous water use efficiency (WUE_i) and decrease rs by 13.9%, 27.2%, and 18.4%, respectively compared to untreated soilless basil plants (Table 2).

3.3. Ion Content Induced by OMP under Different NO₃⁻:Cl⁻ Ratios

Neither NO₃⁻:Cl⁻ ratio nor substrate drench OMP application had a significant effect on Na⁺ concentration (avg. 0.17 g kg⁻¹ dry weight; dw) in leaf tissue (data not shown). Our results demonstrated that NO₃⁻, PO₄³⁻, SO₄²⁻, and Cl⁻ were highly influenced by the interaction NO₃⁻:Cl− ratio × OMP, whereas total N, monovalent (K⁺) and bivalent (Ca²⁺) cations were only affected by the lowest NO₃⁻:Cl⁻ ratio (

Table 3. Effects NO₃⁻:Cl⁻ ratio in the nutrient solution and omeprazole (OMP) application rates on total nitrogen (N), nitrate (NO₃⁻), phosphate (PO₄³⁻), sulphate (SO₄²⁻), potassium (K⁺), calcium (Ca²⁺), magnesium (Mg²⁺), chloride (Cl⁻).

Source of Variance	Total N	NO3−	PO43−	SO42−	K+	Ca2+	Mg2+	Cl−
	(g 100 g−1 DW)	(g kg−1 DW)	(g kg−1 DW)	(g kg−1 DW)	(g kg−1 DW)	(g kg−1 DW)	(g kg−1 DW)	(g kg−1 DW)
NO3−:Cl− (N)
80:20	4.40 a	29.09 a	33.13 a	3.68 a	54.16 a	11.55 a	2.41 ab	5.31 d
60:40	4.05 b	19.82 b	29.78 b	3.30 ab	54.53 a	11.57 a	2.52 a	13.21 c
40:60	3.16 c	6.69 c	21.77 c	2.93 b	54.92 a	12.37 a	2.29 b	26.58 b
20:80	2.19 d	0.92 d	13.66 d	2.35 c	43.26 b	10.15 b	2.39 ab	36.80 a
	***	***	***	***	***	**	ns	***
Omeprazole (μM) (OMP)
0	3.37	11.76	19.86	2.50	43.50	10.34	2.07	24.44
10	3.53	16.51	29.31	3.63	59.93	12.48	2.74	16.51
t-test	0.674	0.339	0.012	0.001	0.001	0.001	0.001	0.048
N × O
80:20 × 0 μM OMP	4.34	24.34 b	26.03 b	2.86 bc	44.59	10.28	2.02	6.73 de
60:40 × 0 μM OMP	4.03	15.56 c	23.25 bc	2.51 cd	44.37	10.76	2.22	16.57 c
40:60 × 0 μM OMP	3.12	6.13 d	18.85 cd	2.53 cd	47.95	11.00	1.98	32.61 b
20:80 × 0 μM OMP	2.02	0.99 e	11.31 e	2.10 d	37.10	9.32	2.04	41.85 a
80:20 × 10 μM OMP	4.47	33.84 a	40.23 a	4.50 a	63.74	12.82	2.80	3.90 e
60:40 × 10 μM OMP	4.08	24.08 b	36.31 a	4.08 a	64.68	12.38	2.81	9.85 d
40:60 × 10 μM OMP	3.21	7.26 d	24.70 b	3.33 b	61.88	13.75	2.60	20.55 c
20:80 × 10 μM OMP	2.37	0.85 e	16.00 d	2.60 cd	49.42	10.97	2.74	31.75 b
	ns	**	*	*	ns	ns	ns	*

ns, *, **, *** not significant or significant at p ≤ 0.05, 0.01 or 0.001, respectively. Different letters within each column indicate significant differences according to Duncan’s multiple-range test (p = 0.05). Omeprazole application rates are compared according to Student’s t-test.

). The N concentration in leaves decreased linearly with increasing Cl⁻ in the nutrient solution, whereas the detrimental effect of chloride salinity on K⁺ and Ca²⁺ was only observed under severe chloride concentration (20:80 treatment; Table 3). Concerning the nitrate, phosphate and sulphate concentration a NO₃⁻:Cl⁻ ratio-dependent response to OMP application was observed with NO3−, PO43−, and SO42− concentration being the highest in OMP-treated plants grown at 80:20 (for nitrate) and at both 80:20 and 60:40 ratios (for phosphate and sulphate) (Table 3). Interestingly, the substrate drench application of 10 µM OMP effectively reduced the toxic anion (Cl⁻) accumulation in leaf tissue under Cl⁻ stress conditions (60:40, 40:60 and 20:80 NO₃⁻, PO₄³⁻, SO₄²⁻ ratios) but not under control conditions (80:20 NO₃⁻, PO₄³⁻, SO₄²⁻ ratio; Table 3). Under Cl⁻ stress conditions OMP-treated basil plants significantly reduced the accumulation of Cl⁻ in the aerial part in a dose-dependent manner: −40.5%, −36.9%, and −24.0% in response to 60:40, 40:60, and 20:80 NO₃⁻:Cl⁻ ratio, respectively (Table 3).

3.4. Heat Map Analysis

The aggregated data heat-map analysis (Figure 2) identified two main clusters corresponding to the 20:80 × 10 µM OMP and 20:80 and 40:60 without OMP on the left and all the other treatments on the right. This indicated that the NO₃⁻:Cl⁻ ratio of nutrient solution was the main clustering factor responsible for different effects. Indeed, two separated sub-clusters could be defined under both the first and the second clusters which illustrated the NO₃⁻:Cl⁻ ratio × OMP interaction. In particular, the OMP application at 20:80 ratio subcluster separated from that with 20:80 and 40:60 ratios without OMP because of the higher WUEi and Mg²⁺ (in common with all other OMP Treatments), lower E, but higher A_CO2. While 80:20 and 60:40 ratios without OMP separated from 80:20, 60:40, and 40:60 ones plus OMP because of their higher dry shoot and root biomass, K⁺ and Ca²⁺. In particular, 80:20 and 60:40 NO₃⁻:Cl⁻ ratios plus OMP showed the highest A_CO2, a*, PO₄³⁻, SO₄²⁻, SPAD index, fresh yield and leaf number. These latter treatments showed also in common with the treatment 80:20 ratio without OMP the highest total N, NO₃⁻, and LN, but the lowest Cl⁻ content. On the other hand, the 20:80 treatment without OMP showed the lowest A_CO2, LA, SPAD, dry shoot and root biomass, K⁺, Ca²⁺, and WUEi (Figure 2).

A different heat map analysis evaluating the changes in the analyzed parameters in dependence on the NO₃⁻:Cl⁻ ratios in the nutrient solution (Figure 3A) and OMP application (Figure 3B) separately was also performed. The decrease of NO₃⁻:Cl⁻ ratio compared to the highest N ratio, showed a clear increase of Cl⁻, b*, L*, and r_s and a decrease of NO₃⁻, total N, fresh yield, leaf area (LA), leaf number (LN), PO₄³⁻, root-to-shoot (R/S) ratio, dry shoot and root biomass, and SO₄²⁻. However, OMP application improved dry root biomass, R/S, PO₄³⁻, Mg²⁺, K⁺ compared to all the respective controls, while Cl⁻ decreased, even if not significantly at 80:20 ratio. Only at 80:20 and 60:40 N, OMP increased NO₃⁻ and SO₄²⁻, while total N decreased. Finally, OMP application increased WUEi and A_CO2 but only at 20:80 ratio.

3.5. Principal Component Analysis

The score plot and loading matrix based on the first and second principal components PC1 and PC2 are reported in Figure 4. The first two PCs were associated with eigen values higher than 1 and explained 90.7% of the cumulative variance, with PC1 and PC2 accounting for 74.9% and 15.8%, respectively (data not shown). PC1 was strongly and positively correlated to morphometric traits (leaf number and area, fresh yield, shoot and root biomass, and R/S ratio), SPAD index and A_CO2 as well as the ion content in leaf tissue (N, NO₃⁻, PO₄³⁻, SO₄²⁻, K⁺, and Ca²⁺); and negatively correlated with leaf color parameters (L* and b*), leaf dry matter percentage, Cl⁻ concentration and r_s. In addition, PC2 was positively associated with Mg²⁺ and WUE_i and negatively correlated with transpiration rate (E). Furthermore, based on the loading matrix, our PCA indicated that variation in above and below ground biomass were most closely aligned with A_CO2, while variation in nitrate content was not correlated to leaf dry matter (DM) percentage (Figure 4).

Moreover, the score plot derived from the PCA clearly divided the OMP application along PC2 with substrate drench OMP-treated plants (except for 80:20 NO₃⁻:Cl⁻ ratio) on the positive side of the second PCs. For instance, basil plants treated with 10 µM OMP were characterized by improved SPAD index and fresh yield (at 80:20 NO₃⁻:Cl⁻ ratio) and higher shoot and root biomass, nutritional, physiological status, and photosynthetic performance (at 60:40 and 40:60 NO₃⁻:Cl⁻ ratio). Finally, the untreated basil plants especially those grown under moderate to severe Cl⁻ stress in the nutrient solution (40:60 and 20:80 NO₃⁻:Cl⁻ ratio) were characterized by the lowest agronomic performance due to the high stomatal resistance and accumulation of toxic ions (Cl⁻ ) in leaf tissue (Figure 4).

4. Discussion

Herbaceous species can be more sensitive to chloride than sodium, with critical levels of chloride varying between 4 and 7 mg g⁻¹ fresh weight (FW) [54]. Mild to moderate concentrations of CaCl₂ can induce ion imbalance and hyperosmotic stress even more severe than NaCl in different horticultural species, reducing plant growth and yield [55,56,57]. This was demonstrated in the present experiment in which basil plants were submitted to a factorial combination of four ratios of nitrate (NO₃⁻) to chloride (Cl⁻) (80:20, 60:40, 40:60, or 20:80).

Significant decrease in morphological traits (e.g., number of leaves, total leaf area and R/S ratio) was noted under severe chloride treatment (20:80 NO₃⁻:Cl⁻ ratio), which, in turn, reduced the fresh yield. Stomatal resistance (r_s) of basil plants increased with the decrease of NO₃⁻:Cl⁻ ratio in the nutrient solution while transpiration (E) was linearly reduced, affecting net CO₂ assimilation rate (A_CO2). Accordingly, Geilfus and co-workers [39] proved that the exposure of Vicia faba plant roots to high Cl⁻ levels could induce a long-distance signal causing leaf apoplastic pH alkalinization, leaf ABA redistribution, stomata closure, and therefore an indirect inhibition of photosynthesis.

NO₃⁻, PO₄³⁻, and SO₄²⁻ were severely reduced by the decrease of NO₃⁻:Cl⁻ ratio while Cl⁻ strongly increased. Chloride uses the same anion channels used by nitrate [58], specifically interfering with nitrate uptake, transport and loading in the root xylem [59]. This implies that nitrate transport to leaves decreases, while excess chloride increases being also less controlled compared to that of sodium [1,56].

To further worsen the chloride salinity impact, there is the lower capacity to exclude chloride than sodium from the leaf blades [1] and the scarce basipetal re-translocation of this ion from leaves to roots via the phloem compared to that of sodium [39,60]. The chloride-dependent reduction of nitrate levels in the cells can indirectly downregulate nitrate uptake by lowering nitrate internal demand [61]. In fact, nitrate in the cell induces the expression and the transcription of genes involved in nitrate assimilation and transport, including high affinity nitrate transporters and nitrate reductase genes, in addition to genes involved in energy and carbon metabolism [62,63,64]. Moreover, nitrate deficiency indirectly accelerates yellowing and senescence of older leaves, since it induces proteolysis of plastidial proteins and amino acid remobilization to young leaves ([65] and references therein). Accordingly, the increase of chloride in basil leaves caused a decrease of photosynthetic pigments as proved by the decrease of the SPAD index and a lighter color of basil leaves (higher L*).

The negative effects of chloride were mitigated by the application of OMP, with a lower beneficial effect of OMP in the treatment 20:80 NO₃⁻:Cl⁻ ratio. In particular, plants growth at 80:20 and 60:40 NO₃⁻:Cl⁻ ratios and treated with OMP showed the highest fresh yield, shoot and root biomass; while those grown with 40:60 NO₃⁻:Cl⁻ ratio and treated with 10 µM OMP did not show a decrease of fresh yield compared to the control without OMP. OMP also determined an increase of the root to shoot ratio (R/S) which is supposed to be a trait of salt tolerance [66].

The treatments with OMP, with the exception of that at 20:80 NO₃⁻:Cl⁻ ratio, kept unchanged the leaf area and number and increased and/or preserved SPAD index, A_CO2 and instantaneous water use efficiency (WUE_i) at levels comparable to those of 80:20 ratio without OMP while decreasing r_s. Probably OMP was able to preserve photosynthetic apparatus and transpiration processes because of the higher nitrate and potassium content, and lower chloride concentration present in basil leaves at 80:20, 60:40, and 40:60 NO₃⁻:Cl⁻ ratios. Cirillo and co-workers [25], by using OMP to ameliorate salinity toxicity in the salt sensitive basil genotype Napoletano, ascribed the beneficial effect of this small bioactive molecule to the higher acquired capacity to exclude chloride from the cytosol of salt stressed cells. However, OMP more than increasing the ability of plant to better compartmentalize chloride in vacuole or exclude it in the apoplast of basil leaf cells, is able to limit the access of chloride to the above ground parts of the plant, as inferred by the lower Cl⁻ content in the leaf in all the NO₃⁻:Cl⁻ treatments with the exception of the 20:80 one. Chloride exclusion is considered the main mechanism contributing to salt tolerance in soybean [67], while, on the contrary, its accumulation in soybean leaves is correlated with decreased transpiration and photosynthesis, reduced crop quality and yield, and finally plant death ([68] and references therein).

The key step of chloride transport and accumulation in the shoot, which antagonizes the nitrate transfer to the shoot, is the loading of chloride from the root stelar symplast to xylem apoplast, which is regulated/inhibited by the stress-induced abscisic acid (ABA) and cytosolic Ca²⁺ [68,69]. As suggested by Rouphael et al. [23] in tomato plants treated with OMP, this small bioactive molecule can trigger signal transduction pathways mediated by endogenous phytohormones or calcium, or in this case induce the synthesis of hormones (i.e., ABA), which can activate sub-traits that confer chloride salinity tolerance.

ABA has no effect on chloride uptake by roots or its compartmentalization in root vacuole ([68] and references therein). However, AtNPF2.5, a homolog of AtNPF2.4 belonging to the root nitrate excretion transporter subfamily (NAXT) localized to the plasma membrane of root cortical cells, has been suggested to encode a transporter which mediates Cl⁻ excretion from the roots under salinity and is also able to transport ABA [70]. OMP could be therefore involved in the regulation of the expression of this NAXT type gene exerting an action of chloride extrusion from root cells in presence of ABA.

Moreover, it has been proved in Arabidopsis that a single missense mutation in CLCa, a member of the family of anion transporters CLC implicated in the vacuolar storage of NO₃⁻, can change the selectivity of the channel from NO₃⁻ to Cl⁻ determining the Cl⁻ compartmentalization in the root vacuoles and consequent lower loading to leaves [71]. OMP could be also involved in this kind of specific epigenetic modification implying the compartmentalization of chloride into vacuoles of root cells thus improving basil tolerance to salinity.

Whatever the reason for reducing the loading of Cl⁻ to shoot, this generates an increase of NO₃⁻ and even more of potassium in the leaves. An outwardly rectifying non-selective cation channel (NORC) identified in barley roots xylem parenchyma cells by Wegner and Raschke [72] could be responsible for this phenomenon. The channel, activated by membrane depolarization, possibly due to active Cl⁻ extrusion or compartmentalization in the vacuole, is permeable to the Cl⁻ and NO₃⁻ but about twice more is permeable to cations [73]. This channel could be responsible for the simultaneous passive non-selective transport/loading of NO₃⁻ and K⁺ to shoot. This could also explain the higher K⁺ content in OMP treated plants than 80:20 ratio without OMP, responsible for the increase in leaf area/expansion and fresh yield. Finally, OMP was no more able to exert a positive effect on basil tolerance to Cl⁻ when very severe Cl⁻ conditions where applied, 20:80 NO₃⁻:Cl⁻ ratio.

5. Conclusions

Chloride toxicity, that is exerted at relatively low concentrations starting from 4–7 mg g⁻¹ in glycophytes, can be more dangerous than sodium because its transport is less controlled. Notwithstanding, the study of chloride salinity has been almost neglected compared to that of sodium. OMP seems responsible for control of chloride transport and exclusion from shoots improving salt tolerance in basil. The fact that it limits chloride loading to shoot while favoring those of nitrate, potassium, and other beneficial ions, boosts photosynthetic activity and leaf metabolism determining a significant increase in leaf expansion and marketable fresh yield. The mechanisms by which OMP can control/reduce chloride loading to shoot certainly require further molecular study in particular about chloride transporter/excluder channels. However, it is clear that OMP use is absolutely beneficial to plants, and can be useful to unravel the molecular mechanisms involved in chloride salt tolerance.