Amelioration of Olive Tree Indices Related to Salinity Stress via Exogenous Administration of Amino Acid Content: Real Agronomic Effectiveness or Mechanistic Restoration Only?

Abstract

Salinization of olive orchards constitutes a front-line agronomic challenge for farmers, consumers, and the scientific community as food security, olive logistics, and land use become more unsustainable and problematic. Plantlets of two olive varieties (var. Kalamon and var. Koroneiki) were tested for their performance under soil saline conditions, in which L-methionine, choline-Cl, and L-proline betaine were applied foliarly to alleviate adverse effects. The ‘Kalamon’ variety ameliorated its photosynthetic rates when L-proline betaine and L-methionine were administered at low saline exposure. The stressed varieties achieved higher leaf transpiration rates in the following treatment order: choline-Cl > L-methionine > L-proline betaine. Choline chloride supported stomatal conductance in stressed var. Kalamon olives without this pattern, which was also followed by var. Koroneiki. Supplementation regimes created a mosaic of responses on varietal water use efficiency under stress. The total phenolic content in leaves increased in both varieties after exogenous application only at the highest levels of saline stress. None of the substances applied to olive trees could stand alone as a tool to mitigate salinity stress in order to be recommended as a solid agronomic practice. The residual exploitation of amino acids by the olive orchard microbiome must also be considered as part of an environmentally friendly, integrated strategy to mitigate salinity stress.

1. Introduction

Ancient Greek and Byzantine mosaics and the list of seven species reported in the Hebrew Bible represent an extremely tiny portion of historic proofs of human–olive tree co-existence and evolution in areas around the Mediterranean Sea [1,2,3]. Historical and aesthetic presence were always in balance with the contributions of Olea europaea to human nutrition and medicine; trials with successful results and failures over centuries formed our current knowledge for the cultivation of trees under optimum and stressed conditions [4,5,6,7]. Apart from olive oil, olive fruit, and tree wood uses in everyday life, olive tree biomass is fully exploitable from brick production to decaffeinated products in the brewed beverage market [8,9,10]. Olive varieties ‘Kalamon’ and ‘Koroneiki’ are traditional and entrepreneurial choices for spatial grove establishment throughout all zones of olive tree cultivation; both varieties are able to offer edible products rich in phenolics, flavonoids, and volatile compounds [11,12,13,14,15].

Either the primary (nature-based) or secondary (human activity-based) salinization of soils poses a major threat to agricultural productivity, affecting both irrigated and non-irrigated lands [16,17,18]. No matter the causative origin of soil salinization, salinity is a major stress factor for all plant species, inducing suboptimal physiological functionality and leading to death when the plant becomes intolerant and toxic; major emphasis is given in tree species due their extended exposure to the stress factor, circularly repetitive demand for high yields, and sustainable entrepreneurial performance [19,20,21,22]. Olive tree is a cultivar-dependent plant species for salinity stress tolerance; it can survive when the electrical conductivity values of irrigation water are lower than 5.0 dS/m in moderately tolerant varieties [23,24]. Furthermore, olives carry anatomical, morphological, and biochemical adaptation mechanisms to drought (leaves with hairy and waxy cuticle, increased cell wall thickness, rigidity) and induce the biosynthesis of specialized osmoprotectants, which are also useful when trees are exposed to salinity stress [22]. The induction of physiological, molecular, and biochemical mechanisms in olive trees has recently been reviewed in depth by El Yamani and Cordovilla [25]. Salinity stress-related memory has been documented in olive trees, providing valuable insights into resilient adaptive phenomena (alterations in mRNA activities; induced protein synthesis) [26].

Methionine is essential for olive tree nutrition and physiology and contributes to primary and secondary plant metabolism (Figure 1) [27,28,29]. It is involved in protein synthesis and the production of osmoprotectants, flavonoids, and volatile compounds like methanethiol [25,30,31,32,33]. It is well known that methionine has a hormonal contribution in plant development as ethylene’s precursor molecule [34]. Application of selenium–methionine was found to alleviate (a) organismal drought stress effects in olive trees, securing unaffected qualitative characteristics of olive oil, and (b) heat stress effects on olive pollen, restoring germination rates due to its protective role against induced oxidative mechanisms [35,36]. Foliar treatment of olive orchards with Zn-methionine complex was found to improve the concentration of oleic acid in the delivered olive oil [37]. Two weeks prior to olive fruit harvest, methionine can be applied as a loosening agent, enhancing anthocyanins, carotene, and fruit color without affecting firmness; however, the side effect of preharvest leaf drop is apparent [38]. Synchronous foliar application of methionine with other essential amino acids is capable of increasing leaf content in nitrogen, phosphorus, potassium, and boron, ameliorating fruit size and yield [39]. Mixtures of methionine with oxylipins, phenylalanine, monosaccharides, and inorganic fertilizers sprayed on olive groves resulted in fruits with color uniformity, a high content of specific phenols such as oleocanthal and 3,4-DHPEA-EDA, and low oil spiciness [40]. Application of methionine restored chlorophyll content, photosynthetic efficiency, antioxidant activities, and osmolytes in C4 field crops like maize [41]. On an industrial scale, under a circular economy context, L-methionine can be produced via E. coli or Corynebacterium glutamicum substrate fermentation, where O-acetyl-L-homoserine reacts enzymatically with CH₃SH to form methionine, which thereafter is collected via evaporation, evaporative crystallization, crystal separation, and drying [42,43,44].

In the agri-food sector, choline chloride is used in green extraction processes, from olive waste polyphenol isolation to spent coffee ground nutraceutical delivery (Figure 2) [46,47]. For tree species, administration of choline chloride on mango trees of cv. Succary Abiad reduced fruitlet abscission and increased the weight and sugar content of fruits [48]. In contrast, foliar applications with choline chloride were found to reduce Ca, Cu, Mn, and Zn in peach fruits and the yield of hydroponically cultivated fig trees in non-stressed conditions [49,50]. At the post-harvest stage, the application of choline chloride to litchi fruits delayed pericarp browning, slowed the respiration rate, postponed programmed cell death, and caused an increase in soluble protein content [51]. In bulb plants like lilium, exogenous administration of 100 mg/L choline chloride increased stem length, total leaf number, flower crown diameter, and leaf content in chlorophyll and sucrose [52]. Shoot and root dry weights of Arabidopsis thaliana increased when choline chloride was applied to the nutritive media. Similar growth data were observed for hydroponically cultivated Brassica napus due to increased photosynthetic gene activity [53]. Suspended cultured sycamore cells accumulate exogenously administered choline in vacuoles and incorporate the molecule into the phosphatidylcholine form [54]. Tomato seedlings supplemented with choline chloride reduced Cd intake and alleviated toxicity in polluted areas without altering H₂O₂ levels in leaves and roots [55]. In a different physiological and biochemical route, exogenous application of choline chloride provides antioxidant protection and resilient growth in spinach under chromium exposure, inducing heavy metal accumulation in shoots and roots [56]. Rehmannia glutinosa seedlings pretreated with choline chloride sustained superoxide dismutase and ascorbate peroxidase activities, enhancing leaf proline content as a crucial plant response to drought stress [57]. Similar enzymatic activity, osmolyte concentration trends, and minimal oxidative damage were reported in saline-stressed tomato plants when supplemented with enriched choline chloride organic manure, with a positive influence on fruit quality and yield [58].

Under saline stress, choline restored total lipids, sterols, and phospholipids content, providing a resilient ratio of sterols/phospholipids in wheat roots [59]. However, in the dominant seashore perennial grass species, the sustainable effect of choline chloride on lipid content did not exhibit preferential changes in lipid synthesis for salt tolerance purposes [60]. Endogenously, halophytic species of the Limonium genus are capable of producing choline-originated osmolytes that affect glycine betaine synthesis and the leaf solute potential [61]. Lipid reprogramming upon the increased presence of digalactosyl diacylglycerol, phosphatidylinositol, and phosphatidic acid due to the exogenous application of choline chloride offers optimized osmotic adjustments and chloroplast membrane stability when plants are exposed to extreme saline environments [60]. Microbial management of choline, which is available in the rhizosphere, enhances corn resilience to salinity stress through two potential routes: advancing plant metabolism and modifying the physicochemical environment for the root’s benefit [62]. Low doses of the quaternary ammonium salt, 2,4-D choline, can increase shoot and root growth in soybean [63].

Betaine is commonly found in plants, microorganisms, and animals, with its concentration in olive oil reported to reach 1 µg/g [64,65]. Betaine synthesis in plants is primarily based on choline via two oxidation stages and secondarily on serine through photorespiration and phosphorylation pathways and ethanolamine under abiotic stress conditions (Figure 3) [66,67,68]. Betaine enhances microbial degradation of toxic substrates and supports intermediate stages of phytoremediation schemes in contaminated soil sites [69]. The most studied form of betaine is glycine betaine which provides (a) higher yield at the crop level; (b) antioxidant protection from heavy metals; (c) osmoregulatory, transcriptional and photosynthetic protective role in drought, salinity, heat, and low temperature stress; (d) alleviation mechanisms of extreme pH conditions in the rhizosphere; and (e) hormonal regulation under stress conditions [70,71,72,73,74,75]. The interest in studying betaine is high, and it has also been documented as a priority in the development of genetically modified plants; however, information available for tree species remains extremely low [76]. Glycine betaine was found to be applicably compatible with various inorganic, organic, and microbial substrates like elements K, Zn, Si, Se, chitosan, bacteria, fungi, and algae extracts to provide faster relief under abiotic stress conditions [77,78,79,80,81]. Other less common forms of betaine in plants are β-alaninebetaine, proline betaine, and pipecolic acid betaine, which have also been found to alleviate the effects of saline stress [82,83,84].

In higher plants, proline is synthesized by the glutamate and ornithine pathways, supporting resilience in abiotic stress exposure and securing optimum functionality of plant developmental mechanisms, embryo development, flowering time, and fertility (Figure 4) [85]. Among other properties, proline plays a significant role in plant cell wall synthesis and topological signaling, drought stress, regulation of root hair development, root biomass, and sensitivity of roots to abscisic acid [86]. The intermediate product of proline biosynthesis and catabolism, pyrroline-5-carboxylate, confers (a) pollen fertility, as reported in Petunia spp., tomato plants, and Arabidopsis thaliana, and (b) plant resistance to several strains of Pseudomonas syringae [87,88]. Some research groups have debated the ability of proline to alleviate abiotic stress due to the numerous failures observed in transgenic plants where proline biosynthesis was overexpressed and the occurrence of adverse effects [89]. Furthermore, excessive proline presence in plant tissues has been proven to induce subcellular organelle oxidative stress and provoke adverse effects, such as (a) structural alterations in chloroplasts resulting in reduced photosynthetic capacity and (b) damage to mitochondrial integrity and subsequently aberrant electron transport, leading to programmed cell death phenomena [90,91].

Herein, due to (a) the available information (Figure 1, Figure 2, Figure 3 and Figure 4), (b) the existing knowledge gap for olive tree (Figure 5), and (c) the fact that salinity is an expansive problem, we examined potential stress alleviation strategies to mild, moderate, and severe exposures using L-methionine, choline chloride, and L-proline betaine nutritional complexes in two olive varieties (var. Koroneiki and var. Kalamon). This work seeks to highlight the biological and agronomical validity of potential benefits and/or weaknesses for olive counter-saline resilience during the summer period, mitigating salinity stress by repetitive amino acid exogenous applications.

2. Materials and Methods

Plant material

Two olive varieties, ‘Koroneiki’ and ‘Kalamon’, with different responses to saline stress, moderately tolerant and tolerant, respectively [92], were used in this experiment. One-year-old olive plants derived from rooted cuttings were grown in 2.5 l pots with peat/perlite (2:1 v/v) substrate under open field conditions at the Agricultural Department of the University of Patras, Greece [N38.366717, E21.476747]. Eighty plants of each cultivar, 85–90 cm tall, were selected, transplanted to 12 l plastic pots with the same substrate, and kept under field conditions. Plants were irrigated every two days; fertigation was applied once every 15 days with N (10 g/L), P (3 g/L), K (9 g/L), Mg (2 g/L), Ca (3 g/L), and B (0.03 g/L) throughout the experimental period (May–September 2023). Mean air temperature and relative humidity values were 19.7 °C (min 12.9 °C, max 31.2 °C) and 55.1–87.5% for May, 24.9 °C (min 16.4 °C, max 34.2 °C) and 51.3–85.4% for June, 29.3 °C (min 19.8 °C, max 40.0 °C) and 43.6–81.5% for July, 27.9 °C (min 19.6 °C, max 39.9 °C) and 43.7–81.3% for August, and 24.5 °C (min 15.7 °C, max 33.2 °C) and 46.5–82.9% for September, respectively.

Amino acid biostimulants

Three formulated products rich in L-methionine (N-P-K 5-20-0 +5% L-methionine), choline-Cl (N-P-K 0-20-7 +7% choline-Cl), and L-proline betaine (N-P-K 25-0-0 +7% betaine +3% L-proline) under the commercial names PHYTOAMINO^®-PN, PHYTOAMINO^®-PK, and PHYTOAMINO^®-N, respectively [Karvelas S.A., Agrinio, Greece], were applied foliarly to alleviate saline stress in separate cultivation lines of the olive plants. Applications of formulations (3% standardized solution, 200 mL per plant) took place with hand pump sprayer one week after the saline irrigation schemes were initiated and, thereafter, every 15 days for 5 months (May to September).

Salinity level

Olive plantlets were exposed to a range of salinity stress levels administered through salinization of irrigation water [pH 7.84, EC: 0.33 dS/m, Na: 4 ppm, Cl: 11 ppm; certified analysis by Institute of Soil and Water Resources, Hellenic Agricultural Organization DEMETER, Thermi-Thessaloniki, GR]. Concentrations of 0 mM, 50 mM, 100 mM, and 150 mM NaCl were applied to stress the experimental lines for both varieties. Once every 2 days, plants were irrigated with saline water until 20% leaching was achieved, ensuring applicable uniformity per salinity stress level throughout the root system [93,94]. To prevent osmotic shock, salt concentration was gradually increased at a rate of 25 mM NaCl per irrigation cycle to achieve the maximum level per treatment.

Plant growth

Olive tree growth was assessed by measuring the shoot length from soil level to shoot tip every 30 days, coupled with calculation of growth increment percentage. Number of lateral shoots formed each month was recorded, and their lengths were measured only once at the end of the experimental period.

Total chlorophyll and anthocyanin content

To determine the total chlorophyll and anthocyanin content, readings were taken using the following handheld devices: (a) a total chlorophyll content meter (CCM-200 Plus, OPTI-SCIENCES, Hudson, NH, USA) and (b) an anthocyanin content meter (ACM-200 Plus, OPTI-SCIENCES, Hudson, NH, USA). Leaves from the upper middle section of the main stem were selected to assess their chlorophyll and anthocyanin content under soil salinity stress for all treatments. Data collection was set between 9:00 and 12:00 a.m. every 30 days from May to September. In total, 25 leaves per treatment and 5 records per leaf were collected.

Physiological parameters

A portable photosynthesis measuring system (LCproSD, ADC BioScientific Ltd., Hoddesdon, Herts, UK) was used for direct measurement of leaf photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (gs), photosynthetically active radiation (PAR), leaf incident radiation (Qleaf), and the ratio of intercellular CO₂ concentration to ambient CO₂ concentration (Cin/Cout). The leaf chamber was configured to cover an exposed leaf area of 1.75 cm² to fit olive leaves. A constant gas flow rate of 200 mL min⁻¹ was maintained within the chamber. The CO₂ concentration was automatically controlled by the LCproSD gas exchange system, while incident radiation and vapor pressure deficit were those of ambient conditions. The chamber temperature was set to 25 °C.

The photosynthetic quantum yield (QY) of leaves was calculated from Pn and Qleaf values according to the following equation [95]:

$$\mathrm{Q}\mathrm{Y}={\displaystyle \frac{\mathrm{P}\mathrm{n} (\mathsf{\mu }\mathrm{m}\mathrm{o}\mathrm{l} {\mathrm{C}\mathrm{O}}_2)}{\mathrm{Q}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{f} (\mathsf{\mu }\mathrm{m}\mathrm{o}\mathrm{l} \mathrm{p}\mathrm{h}\mathrm{o}\mathrm{t}\mathrm{o}\mathrm{n}\mathrm{s})}}$$

(1)

The instantaneous water use efficiency (WUE) of leaves was determined from Pn and E values given from photosynthetic device using the following equation [96]:

$$\mathrm{W}\mathrm{U}\mathrm{E}={\displaystyle \frac{\mathrm{P}\mathrm{n} (\mathsf{\mu }\mathrm{m}\mathrm{o}\mathrm{l} {\mathrm{C}\mathrm{O}}_2)}{\mathrm{E} (\mathrm{m}\mathrm{m}\mathrm{o}\mathrm{l} {\mathrm{H}}_2\mathrm{O})}}$$

(2)

Twenty-five fully expanded and sun-exposed leaves per treatment (five leaves per plant) distributed on the upper end of annual shoots were used for physiological parameter assessment of leaves. Three records per leaf were taken from the photosynthetic unit at 30 s intervals. All measurements took place in the morning hours (9:00–12:00 a.m.) at four periods during the growth season (June, July, August, and September).

Total phenols and antioxidant activity

Leaf extract: Leaf samples (50 leaves per treatment) were collected in early September. The leaves were washed with distilled water, dried at 40 °C, and crushed into 0.2 mm pieces with grinding mill (KINEMATICA, POLYMIX PX-MFC 90 D Blade Grinding Mill, Malters, LU, Switzerland). A total of 0.5 g of ground leaves from each sample was added into a screw-capped plastic tube containing 20 mL of methanol (80:20 v/v methanol: water), covered with aluminum foil to protect from light exposure, and left in shaker stirrer (170 rpm) for 24 h at room temperature for maceration. The mixture was centrifuged at 5000 rpm for 10 min, and the supernatant was collected [97].

Total phenolic content: The total phenolic content of the leaf extracts was determined using the Folin–Ciocalteau method [98] with gallic acid (GAE) as the reference standard. For each sample, 0.2 mL of extract, 9.8 mL of distilled water, and 0.75 mL of F-C reagent were added to a 16 mL test tube. After 3 min, 0.25 mL of 20% aqueous sodium carbonate (Na₂CO₃) solution was added, and the reaction mixture was incubated in the dark at room temperature. After 60 min, absorbance was measured at 750 nm using a UV-VIS spectrophotometer (Shimadzu UV 1900i, Kyoto, Japan). The total phenolic content of the samples was calculated using the linear equation obtained from the gallic acid standard curve (y = 0.0024x + 0.0108, R² = 0.9994), and the results were expressed as mg of gallic acid (GAE) equivalent per gram of dry weight of leaves (mg GAE/g d.w.).

Ferric Reducing Antioxidant Power (FRAP) Assay: The FRAP assay was carried out to determine the total antioxidant capacity of the samples following the method described by Benzie and Strain [99]. A fresh FRAP reagent was prepared just before use by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM HCl, and 20 mM FeCl₃.6H₂O in a ratio of 10:1:1. For the assay, 100 μL of olive leaf extract was mixed with 900 μL of distilled H₂O and 3 mL FRAP reagent. The mixture was incubated in a water bath at 37 °C for 4 min, and the absorbance was immediately measured at 593 nm using a UV–VIS spectrophotometer (Shimadzu UV 1900i, Kyoto, Japan). For the calibration curve, 5 aqueous solutions of FeSO₄.7H₂O with concentrations ranging from 100 to 1000 mg/L were used. The equation (y = 0.0007x + 0.0172, R² = 0.9977) obtained from the linear regression of the standard curve was used to calculate the FRAP value of leaf extracts, and the results were expressed as μmol FeSO₄ per gram of dry weight of leaves (μmol FeSO₄/g d.w.).

Statistical Analysis

A factorial completely randomized design with 32 treatments—2 olive varieties (‘Kalamon’ and ‘Koroneiki’), 4 saline levels (0, 50, 100, 150 mM NaCl), and 4 types of supplementation regimes (L-methionine, choline, L-proline betaine, and non-supplementation)—was used in this study. A minimum of five replicates was used for each value, or otherwise, it was stated.

Data were analyzed using the 95% confidence limits overlap protocol of Sokal and Rohlf [100]. The tables and graphic data were presented as means ± standard error of the mean. An α level of 0.05 was chosen. Prism 8.0 (GraphPad) was used for data and graph presentation. Differences between the types of exogenous supplementation, months, and olive cultivars were assessed (SPSS version 29.0).

3. Results

3.1. Olive Growth Rate, Lateral Number, and Length

Saline-free cultivated ‘Kalamon’ plantlets declined in their growth rates in August (Figure 6); administration of amino acids to plantlets did not further increase their growth status. Under saline conditions (100 mM NaCl), the plantlets exhibited low growth rates, particularly in July and August. None of the amino acids applied to olives raised growth rates above those in non-supplemented experimental lines exposed to saline stress.

‘Koroneiki’ plantlets under a salt-free soil environment exhibited no significant differences in growth rates compared to lines with foliar amino acid supplementation. Salinity did not affect growth rates with the exception of 150 mM NaCl levels, where extremely low growth was recorded in July (Figure 6). None of the amino acid treatments ameliorated growth rates in ‘Koroneiki’ experimental lines, without being dependent on the levels of applied stress.

Salinity and amino acid supplementation did not affect the ‘Koroneiki’ variety in terms of the number of laterals. A similar data pattern was also recorded for ‘Kalamon’ plantlets (Figure 7), where no amino acid supplementation ameliorated the values of this parameter. In contrast, foliar application of choline-Cl on saline-free ‘Kalamon’ olives resulted in the lowest recorded number of laterals among all experimental lines.

Lateral length of var. Koroneiki plantlets declined by 53% under 150 mM NaCl soil conditions. Amino acid supplementation did not positively alter this data pattern. Application of L-methionine and L-proline betaine under salt-free soil conditions suppressed the lateral length by 57.79% and 70.18%, respectively. Lateral length was not affected by amino acid supplementation or levels of saline stress in olive var. Kalamon. Non-amino acid-treated ‘Kalamon’ plantlets exhibited steadily lower lateral lengths than ‘Koroneiki’ plantlets without a pattern of dependence on the levels of applied saline stress (Figure 8).

3.2. Photosynthetic Rates

Under unchallenged saline conditions, non-supplemented ‘Kalamon’ olives during the period of June–September provided photosynthetic rates equal to those of the supplemented ones, with the exception of June’s foliar application of L-proline betaine and August’s foliar application of choline-Cl, where performance was poorer. For all unchallenged to saline experimental lines, similar patterns of photosynthetic rates were recorded; from June to July, an apparent transition towards the highest values of photosynthetic rates was mostly observed (22.38–23.19%), followed by a sharp decline in August (46.87–68.07%) and September (61.27–80.73%) (

Table 1. Leaf photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (gs), water use efficiency (WUE), quantum yield (QY), and the ratio of intercellular CO₂ concentration to ambient CO₂ concentration (Cin/Cout) of olive var. Kalamon plantlets during the experimental period (June–September) ⁽¹⁾.

Saline Soil Stress (NaCl)	Treat.(2)	Pn (μmol m−2.s−1)	E (mmol m−2.s−1)	gs (mmol m−2.s−1)	WUE (μmol CO2/mmol H2O)	QY	Cin/Cout
June
0 mM	NS	11.44 ± 0.03ab,a,a	1.73 ± 0.02a,a,a	0.146 ± 0.003a,a,a	6.60 ± 0.07a,a,a	0.70 ± 0.02a,a,a	0.58 ± 0.01a,a,a
	PN	9.87 ± 0.04a,a,a	1.16 ± 0.02a,b,a	0.090 ± 0.000a,b,a	8.49 ± 0.18a,b,a	0.88 ± 0.03a,b,a	0.48 ± 0.01a,a,a
	PK	11.43 ± 0.12a,a,a	1.64 ± 0.01a,a,a	0.143 ± 0.003a,a,a	6.95 ± 0.10a,ab,a	0.78 ± 0.03a,ab,a	0.57 ± 0.01a,a,a
	N	6.88 ± 0.77a,b,ab	1.02 ± 0.17ab,b,ab	0.073 ± 0.018ac,b,ab	6.87 ± 0.67ab,a,a	0.45 ± 0.05a,c,a	0.55 ± 0.05a,a,a
50 mM	NS	6.54 ± 0.48a,a,b	0.99 ± 0.06a,a,b	0.056 ±0.006a,a,b	6.58 ± 0.31a,a,a	0.31 ± 0.01a,a,b	0.50 ± 0.03a,a,a
	PN	6.78 ± 0.15a,a,b	0.89 ± 0.00ab,a,b	0.063 ± 0.003ab,a,b	7.71 ± 0.06a,b,a	0.40 ± 0.01a,bc,b	0.47 ± 0.00a,a,a
	PK	6.47 ± 0.06a,a,b	0.92 ± 0.03a,a,b	0.060 ± 0.000a,a,b	7.02 ± 0.18a,c,a	0.38 ± 0.01a,c,b	0.51 ± 0.01bc,a,b
	N	9.50 ± 0.09a,b,b	1.58 ± 0.01a,b,a	0.110 ± 0.000a,b,a	6.00 ± 0.05a,a,a	0.48 ± 0.00a,d,a	0.56 ± 0.01a,a,a
100 mM	NS	4.14 ± 0.13a,a,c	0.77 ± 0.04a,a,bc	0.046 ± 0.003a,a,bc	5.37 ± 0.12a,a,b	0.24 ± 0.02bc,a,c	0.59 ± 0.00a,a,a
	PN	5.98 ± 0.30a,a,b	0.83 ± 0.03a,a,b	0.046 ± 0.003a,a,c	7.44 ± 0.55a,a,a	0.35 ± 0.03a,a,b	0.46 ± 0.05a,a,a
	PK	6.07 ± 0.15a,a,b	0.87 ± 0.05a,a,b	0.050 ± 0.005a,a,b	6.99 ± 0.30a,a,a	0.36 ± 0.01a,a,bc	0.48 ± 0.02ab,a,b
	N	5.98 ± 1.14a,a,a	1.09 ± 0.20a,a,ab	0.070 ± 0.015a,a,ab	5.61 ± 0.75a,a,a	0.44 ± 0.14a,a,a	0.59 ± 0.05a,a,a
150 mM	NS	3.11 ± 0.13ab,a,c	0.59 ± 0.06a,a,c	0.030 ± 0.005ab,a,c	5.30 ± 0.37a,a,b	0.18 ± 0.01c,a,d	0.58 ± 0.03a,a,a
	PN	3.95 ± 0.10a,a,c	0.54 ± 0.02a,a,c	0.030 ± 0.000ab,a,d	7.29 ± 0.27a,b,a	0.23 ± 0.01a,bc,c	0.44 ± 0.04a,b,a
	PK	5.82 ± 0.28a,b,b	0.91 ± 0.06a,b,b	0.053 ± 0.006a,b,b	6.39 ± 0.12a,ab,a	0.30 ± 0.01a,cd,c	0.51 ± 0.01ab,ab,b
	N	5.27 ± 0.22a,b,a	0.78 ± 0.03a,ab,b	0.043 ± 0.003a,ab,b	6.76 ± 0.03a,b,a	0.32 ± 0.03a,d,a	0.48 ± 0.01ab,ab,a
July
0 mM	NS	14.74 ± 1.72a,a,a	1.55 ± 0.11a,a,a	0.153 ± 0.017a,a,a	9.44 ± 0.43b,a,a	0.78 ± 0.11a,a,a	0.48 ± 0.02a,a,a
	PN	12.85 ± 0.07b,a,a	1.53 ± 0.03b,a,a	0.163 ± 0.006b,a,a	8.37 ± 0.14a,a,a	0.70 ± 0.03b,a,a	0.56 ± 0.01a,a,a
	PK	11.37 ± 0.67a,a,a	1.39 ± 0.03a,a,a	0.146 ± 0.003a,a,a	8.19 ± 0.68a,a,a	0.65 ± 0.052,a,a	0.58 ± 0.033,a,a
	N	10.64 ± 0.05b,a,a	1.36 ± 0.09a,a,a	0.123 ± 0.008a,a,a	7.86 ± 0.57b,a,a	0.54 ± 0.00a,a,a	0.55 ± 0.023,a,a
50 mM	NS	6.19 ± 0.54a,a,b	0.94 ± 0.00a,a,b	0.066 ± 0.003a,a,b	6.54 ± 0.56a,a,b	0.35 ± 0.04a,a,b	0.57 ± 0.03a,a,a
	PN	9.79 ± 0.57b,b,b	1.34 ± 0.24a,a,a	0.116 ± 0.023a,a,a	7.73 ± 1.21a,a,a	0.61 ± 0.01b,b,a	0.55 ± 0.05a,a,a
	PK	6.78 ± 0.54a,b,b	1.16 ± 0.08a,a,a	0.090 ± 0.010b,a,b	5.83 ± 0.02b,a,bc	0.33 ± 0.02a,a,b	0.63 ± 0.00a,a,a
	N	5.99 ± 0.03b,b,b	0.97 ± 0.02b,a,b	0.070± 0.000b,a,b	6.14 ± 0.16a,a,b	0.30 ± 0.01b,a,b	0.59 ± 0.01a,a,a
100 mM	NS	4.46 ± 0.13a,a,b	0.62 ± 0.06ab,a,b	0.040 ± 0.000a,a,b	7.26 ± 0.53b,a,b	0.34 ± 0.04c,a,b	0.57 ± 0.01a,a,a
	PN	6.95 ± 0.26a,c,c	1.22 ± 0.07b,b,ab	0.070 ± 0.005a,b,b	5.68 ± 0.18a,bc,a	0.37 ± 0.01a,a,b	0.53 ± 0.02a,ab,a
	PK	6.17 ± 0.29a,bc,b	1.17 ± 0.05b,b,a	0.076 ± 0.003b,b,b	5.25 ± 0.18a,c,c	0.40 ± 0.05a,a,b	0.61 ± 0.01a,a,a
	N	5.56 ± 0.28a,ab,bc	0.85 ± 0.05ab,a,b	0.046 ± 0.003ab,a,c	6.51 ± 0.10a,ab,ab	0.31 ± 0.01a,a,b	0.48 ± 0.01a,b,b
150 mM	NS	3.48 ± 0.49a,a,b	0.66 ± 0.08a,a,b	0.033 ± 0.003a,a,b	5.27 ± 0.14a,a,b	0.17 ± 0.02bc,a,b	0.56 ± 0.01a,a,a
	PN	3.89 ± 0.42ab,a,d	0.67 ± 0.13a,a,b	0.036 ± 0.006a,a,b	5.99 ± 0.51ac,ab,a	0.21 ± 0.03ab,ab,b	0.57 ± 0.03a,a,a
	PK	5.24 ± 0.38a,a,b	0.73 ± 0.03ab,a,b	0.036 ± 0.003ab,a,c	7.17 ± 0.29a,b,ab	0.28 ± 0.02a,b,b	0.39 ± 0.02b,b,b
	N	4.94 ± 0.24ac,a,c	0.89 ± 0.03a,a,b	0.046 ± 0.003a,a,c	5.51 ± 0.16a,a,b	0.27 ± 0.01b,b,b	0.54 ± 0.01bc,a,ab
August
0 mM	NS	7.83 ± 1.04b,a,a	1.47 ± 0.26a,a,a	0.070 ± 0.015b,a,a	5.43 ± 0.38a,ab,a	0.47 ± 0.06b,a,a	0.48 ± 0.04a,a,ab
	PN	6.81 ± 0.15c,ab,a	1.21 ± 0.07a,a,a	0.060 ± 0.005c,a,a	5.66 ± 0.23b,a,a	0.49 ± 0.03c,a,a	0.50 ± 0.02a,ab,a
	PK	3.63 ± 1.10b,b,a	0.86 ± 0.17b,a,a	0.040 ± 0.010b,a,a	4.05 ± 0.41b,b,a	0.28 ± 0.09b,a,a	0.63 ± 0.03a,b,a
	N	6.29 ± 0.86ac,ab,a	1.16 ± 0.12ab,a,a	0.060 ± 0.010c,a,a	5.37 ± 0.34a,ab,a	0.53 ± 0.04a,a,a	0.55 ± 0.02a,ab,a
50 mM	NS	3.35 ± 0.55b,ab,b	0.71 ± 0.05b,ab,b	0.026 ± 0.003b,a,b	4.62 ± 0.40a,a,a	0.19 ± 0.03c,a,b	0.54 ± 0.05a,a,ab
	PN	2.86 ± 0.25c,b,b	0.63 ± 0.12bc,b,b	0.020 ± 0.005b,a,b	4.79 ± 0.77a,a,a	0.17 ± 0.01c,a,b	0.52 ± 0.08a,a,a
	PK	5.86 ± 0.54a,a,a	0.98 ± 0.03a,a,a	0.043 ± 0.003ac,b,a	5.54 ±0.28b,a,a	0.35 ± 0.02a,b,a	0.47 ± 0.02b,a,a
	N	2.98 ± 0.77c,b,b	0.56 ± 0.03c,b,b	0.023 ± 0.003c,a,b	5.28 ± 1.23a,a,a	0.17 ± 0.05c,a,b	0.51 ± 0.09a,a,a
100 mM	NS	2.35 ± 0.25b,a,b	0.40 ± 0.02bc,a,b	0.023 ± 0.003b,a,b	5.76 ± 0.31ab,a,a	0.13 ± 0.01ab,a,b	0.63 ± 0.02a,a,a
	PN	2.11 ± 0.77b,a,b	0.35 ± 0.06c,a,b	0.020 ± 0.005c,a,b	6.79 ± 2.89a,a,a	0.13 ± 0.05b,a,b	0.60 ± 0.16a,a,a
	PK	3.30 ± 0.79b,a,a	0.47 ± 0.04c,a,b	0.030± 0.000c,a,a	6.84 ± 1.08a,a,a	0.20 ± 0.04b,a,a	0.59 ± 0.07ab,a,a
	N	2.68 ± 0.53a,a,b	0.28 ± 0.03b,a,b	0.016 ± 0.003b,a,b	9.36 ± 0.95b,a,b	0.15 ± 0.03a,a,b	0.44 ± 0.05a,a,a
150 mM	NS	2.15 ± 0.22bc,a,b	0.21 ± 0.02b,a,b	0.010 ± 0.000c,a,b	10.00 ± 0.64b,a,b	0.14 ± 0.02ab,a,b	0.43 ± 0.03a,a,b
	PN	3.15 ± 0.21b,b,b	0.32 ± 0.01a,b,b	0.020 ± 0.000ab,ab,b	9.65 ± 0.35b,a,a	0.16 ± 0.01bc,a,b	0.26 ± 0.01b,b,a
	PK	3.04 ± 0.08b,b,a	0.49 ± 0.04bc,c,b	0.033 ± 0.003bc,cd,a	6.22 ± 0.38a,b,a	0.16 ± 0.01b,a,a	0.62 ± 0.03a,c,a
	N	2.75 ± 0.15c,ab,b	0.40 ± 0.01b,bc,b	0.023 ± 0.003b,bd,b	6.79 ± 0.52a,b,a	0.15 ± 0.01c,a,b	0.59 ± 0.03c,c,a
September
0 mM	NS	2.84 ± 0.50c,a,a	0.68 ± 0.14b,a,a	0.033 ± 0.008b,a,a	4.47 ± 1.37a,a,a	0.14 ± 0.02c,a,a	0.62 ± 0.11a,a,a
	PN	4.33 ± 0.17d,a,a	0.57 ± 0.08c,a,a	0.033 ± 0.003d,a,a	7.78 ± 0.92a,a,a	0.22 ± 0.01d,a,a	0.44 ± 0.05a,a,a
	PK	2.89 ± 0.34b,a,a	0.39 ± 0.04c,a,a	0.016 ± 0.003b,a,a	7.35 ± 0.31a,a,a	0.15 ± 0.02b,a,a	0.43 ± 0.03b,a,a
	N	4.12 ± 0.65c,a,a	0.62 ± 0.10b,a,a	0.033 ± 0.008c,a,a	6.68 ± 0.43ab,a,a	0.21 ± 0.04b,a,a	0.50 ± 0.02a,a,a
50 mM	NS	1.26 ± 0.10c,a,a	0.21 ± 0.01c,a,b	0.010 ± 0.000b,a,b	5.97 ± 0.70a,a,a	0.07 ± 0.01d,a,a	0.50 ± 0.05a,a,a
	PN	2.67 ± 0.28c,a,a	0.43 ± 0.04c,a,a	0.016 ± 0.003b,a,a	6.17 ± 0.04a,a,ab	0.13 ± 0.01c,a,a	0.47 ± 0.01a,a,a
	PK	2.46 ± 0.56b,a,a	0.46 ± 0.10b,a,a	0.020 ± 0.005c,a,a	5.25 ± 0.18b,a,a	0.12 ± 0.03b,a,a	0.55 ± 0.01c,a,a
	N	2.10 ± 0.73c,a,a	0.34 ± 0.08c,a,a	0.013 ± 0.003c,a,a	6.53 ± 1.22a,a,a	0.11 ± 0.03c,a,a	0.52 ± 0.05a,a,a
100 mM	NS	2.42 ± 0.62b,a,a	0.37 ± 0.06c,a,b	0.013 ± 0.003b,a,ab	6.27 ± 0.50ab,a,a	0.12 ± 0.030,a,a	0.44 ± 0.03b,a,a
	PN	2.41 ± 0.86b,a,a	0.37 ± 0.12c,a,a	0.016 ± 0.006c,a,a	6.31 ± 0.28a,a,ab	0.11 ± 0.04b,a,a	0.41 ± 0.02a,a,a
	PK	2.24 ± 0.32b,a,a	0.32 ± 0.02c,a,a	0.010± 0.000d,a,a	6.81 ± 0.63a,a,a	0.11 ± 0.02b,a,a	0.39 ± 0.05b,a,a
	N	2.73 ± 0.99a,a,a	0.49 ± 0.20b,a,a	0.020 ± 0.010b,a,a	5.74 ± 0.38a,a,a	0.13 ± 0.04a,a,a	0.46 ± 0.04a,a,a
150 mM	NS	1.49 ± 0.08c,a,a	0.37 ± 0.06b,a,b	0.013 ± 0.003bc,a,ab	4.30 ± 0.95a,a,a	0.07 ± 0.00a,a,a	0.60 ± 0.08a,a,a
	PN	2.22 ± 0.57b,a,a	0.40 ± 0.08a,a,a	0.016 ± 0.003b,a,a	5.37 ± 0.37c,a,b	0.11 ± 0.02c,ab,a	0.49 ± 0.03a,a,a
	PK	2.76 ± 0.07b,a,a	0.38 ± 0.06c,a,a	0.016 ± 0.003c,a,a	6.86 ± 0.85a,a,a	0.15 ± 0.02b,ab,a	0.38 ± 0.06b,a,a
	N	3.23 ± 0.75c,a,a	0.50 ± 0.10b,a,a	0.020 ± 0.005b,a,a	6.41 ± 0.15a,a,a	0.17 ± 0.03bc,b,a	0.42 ± 0.01a,a,a

⁽¹⁾ The different letters indicate a significant (p < 0.05) difference. The first letter demonstrates differences between months for the same exogenous treatment and level of salinity stress, the second demonstrates differences among exogenous treatments for the same month and level of salinity stress, and the third demonstrates differences for the same exogenous treatment between salinity stress levels for the same monthly period. Data are presented as the mean ± SE of five replicates. ⁽²⁾ Treat: treatment type; NS: non-supplemented olives; PN: L-methionine-supplemented; PK: choline-Cl-supplemented; N: L-proline betaine-supplemented.

).

Saline stress caused a decline in photosynthetic rates in unsupplemented ‘Kalamon’ olives in a stress- and time-dependent manner. During the first month (June), even at the lowest applied stress levels (50 mM NaCl), photosynthesis dropped by 42.83% with higher losses recorded at 150 mM NaCl stress (72.11%). In July, unchallenged saline plantlets exhibited the highest photosynthetic rates; in contrast, salt stress exposure suppressed the rates by 58%, 69.74%, and 76.39% as stress levels in soil increased from mild (50 mM NaCl) to moderate (100 mM NaCl) and severe (150 mM NaCl). A similar trend of losses in August for all levels of NaCl applied to the soil (50, 100, and 150 mM) was recorded at 57.21%, 69.98%, and 72.54%, respectively. The photosynthetic activity in September affected all plantlets; however, no major differences were found to be related to saline stress.

At 50 mM NaCl, soil saline stress foliar supplementation with L-proline betaine in June and L-methionine in July induced greater photosynthetic rates (31.15% and 36.77%, respectively) compared to non-supplemented trees. Amino acid administration on plantlets under increased saline stress conditions (100 mM NaCl) appeared to have a major positive effect in July (19.78–35.82%). Higher photosynthetic rates in comparison to non-treated olives were also recorded for choline-Cl (46.56%) and L-proline betaine (40.98%) in June and for all amino acid treatments in August (21.81–31.74%) for the same levels of saline stress. Soil saline conditions at 150 mM NaCl showed a positive contribution of choline-Cl and L-proline betaine treatments during June and August to the outcome photosynthetic values.

Olive var. Koroneiki without any supplementation lowered its photosynthetic rates from June to September, a pattern that was also observed in all experimental lines. Higher rates were recorded in June for L-proline betaine-treated plantlets (14.56%), in July for L-methionine (14.92%) and choline-Cl (15.61%), and in August for all amino acid-treated plants (22.03–37.31%) (

Table 2. Leaf photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (gs), water use efficiency (WUE), quantum yield (QY), and the ratio of intercellular CO₂ concentration to ambient CO₂ concentration (Cin/Cout) of var. Koroneiki olive plantlets during the experimental period (June–September) ⁽¹⁾.

Saline Stress (NaCl)	Treat.(2)	Pn (μmol m−2.s−1)	E (mmol m−2.s−1)	gs (mmol m−2.s−1)	WUE (μmol CO2/mmol H2O)	QY	Cin/Cout
June
0 mM	NS	10.03 ± 0.18a,ab,a	1.51 ± 0.14a,a,a	0.103 ± 0.012a,a,a	6.72 ± 0.57a,a,a	0.91 ± 0.03a,a,a	0.49 ± 0.05a,a,a
	PN	11.31 ± 0.06a,bc,a	1.64 ± 0.08a,a,a	0.106 ± 0.008a,a,a	6.94 ± 0.39a,a,a	0.89 ± 0.03a,a,a	0.47 ± 0.03a,a,a
	PK	9.08 ± 0.54a,a,a	1.31 ± 0.09a,a,a	0.066 ± 0.003a,b,a	6.93 ± 0.11ab,a,ab	0.45 ± 0.01a,b,a	0.38 ± 0.00a,a,a
	N	11.74 ± 0.18a,c,a	1.57 ± 0.07a,a,a	0.106 ± 0.008a,a,a	7.50 ± 0.26a,a,a	0.59 ± 0.01a,c,a	0.44 ± 0.02a,a,a
50 mM	NS	8.29 ± 0.09a,a,b	1.09 ± 0.02a,a,ab	0.063 ± 0.003a,a,b	7.61 ± 0.06a,a,a	0.45 ± 0.00a,a,bc	0.43 ± 0.01ab,a,a
	PN	9.91 ± 0.05a,b,a	1.28 ± 0.04a,a,b	0.083 ± 0.006a,a,ab	7.73 ± 0.33ab,a,ab	0.54 ± 0.00a,a,b	0.43 ± 0.03a,a,a
	PK	8.01 ± 0.25a,a,a	1.14 ± 0.05a,a,ab	0.066 ± 0.008a,a,a	7.03 ± 0.24ab,a,ab	0.51 ± 0.01a,a,ab	0.45 ± 0.03a,a,a
	N	9.19 ± 0.65a,ab,b	1.25 ± 0.13a,a,ab	0.080 ± 0.011a,a,ab	7.39 ± 0.32a,a,a	0.48 ± 0.04a,a,a	0.44 ± 0.03a,a,a
100 mM	NS	7.42 ± 0.44a,ab,b	0.96 ± 0.06a,a,b	0.060 ± 0.005a,a,b	7.40 ± 0.17a,a,a	0.42 ± 0.03a,a,c	0.44 ± 0.02a,a,a
	PN	7.62 ± 0.68a,ab,b	1.14 ± 0.07a,a,b	0.070 ± 0.005a,a,bc	6.66 ± 0.17a,a,a	0.40 ± 0.03a,a,c	0.43 ± 0.01ab,a,a
	PK	8.83 ± 0.37a,a,a	1.17 ± 0.04a,a,ab	0.070 ± 0.005a,a,a	7.54 ± 0.14a,a,a	0.45 ± 0.03a,a,a	0.41 ± 0.01a,a,a
	N	6.16 ± 0.29a,b,c	0.91 ± 0.05a,a,bc	0.050 ± 0.005a,a,b	6.78 ± 0.42ab,a,a	0.32 ± 0.006a,b,b	0.46 ± 0.03a,a,a
150 mM	NS	7.14 ± 0.30a,ab,b	1.01 ± 0.10a,a,b	0.066 ± 0.008a,a,b	7.13 ± 0.55a,a,a	0.52 ± 0.02a,a,b	0.50 ± 0.04a,a,a
	PN	7.10 ± 0.49a,ab,b	0.80 ± 0.06a,a,c	0.053 ± 0.003a,a,c	8.88 ± 0.09a,b,b	0.560 ± 0.02a,a,b	0.44 ± 0.00a,ab,a
	PK	6.22 ± 0.28a,b,b	0.93 ± 0.01a,a,b	0.053 ± 0.003a,a,a	6.66 ± 0.21b,a,b	0.57 ± 0.04a,a,b	0.55 ± 0.01b,a,b
	N	8.18 ± 0.23a,a,b	0.80 ± 0.04a,a,c	0.053 ± 0.003a,a,b	10.27 ± 0.35a,b,b	0.58 ± 0.04a,a,a	0.35 ± 0.03a,b,a
July
0 mM	NS	7.24 ± 0.04b,a,a	1.20 ± 0.09ab,a,a	0.076 ± 0.008ab,a,a	6.08 ± 0.48a,a,a	0.49 ± 0.02b,ab,a	0.57 ± 0.03a,a,b
	PN	8.51 ±0.30b,b,a	1.42 ± 0.07ab,a,a	0.100 ± 0.005a,a,a	5.98 ± 0.22ab,a,a	0.57 ± 0.03b,b,a	0.58 ± 0.02b,a,bc
	PK	8.58 ± 0.07a,b,a	1.43 ± 0.04ab,a,a	0.096 ± 0.008b,a,a	6.02 ± 0.23bc,a,a	0.56 ± 0.02b,b,a	0.56 ± 0.03b,a,ab
	N	6.78 ± 0.43b,ac,a	1.17 ± 0.09b,a,a	0.083 ± 0.008ab,a,a	5.79 ± 0.08b,a,a	0.44 ± 0.02b,a,a	0.60 ± 0.01bc,a,a
50 mM	NS	6.16 ± 0.01b,ac,b	0.84 ± 0.04b,a,b	0.053 ± 0.003a,a,ab	7.32 ± 0.40a,ab,ab	0.34 ± 0.01b,a,b	0.52 ± 0.03c,ab,ab
	PN	8.49 ± 0.40b,b,a	1.03 ± 0.04b,a,b	0.076 ± 0.003a,a,ab	8.24 ± 0.13ab,b,b	0.45 ± 0.02b,b,ab	0.48 ± 0.01a,a,ab
	PK	5.99 ± 0.56b,c,b	0.88 ± 0.10b,a,b	0.056 ± 0.008a,a,b	6.82 ± 0.18ab,a,a	0.33 ± 0.02b,a,b	0.55 ± 0.01a,ab,ab
	N	6.53 ± 0.45b,a,a	0.97 ± 0.05a,a,a	0.070 ± 0.005a,a,a	6.73 ± 0.22ab,a,ab	0.37 ± 0.03a,ab,a	0.57 ± 0.02b,b,ab
100 mM	NS	4.65 ± 0.26b,a,c	0.57 ± 0.02b,a,c	0.033 ± 0.003bc,a,b	8.11 ± 0.08a,a,b	0.24 ± 0.01b,a,c	0.44 ± 0.01a,a,a
	PN	7.07 ± 0.71ab,b,a	0.92 ± 0.05ab,b,b	0.050 ± 0.005b,a,bc	7.61 ± 0.46a,a,ab	0.38 ± 0.05a,b,bc	0.403± 0.04a,a,a
	PK	5.12 ± 0.16b,a,bc	0.72 ± 0.08b,ab,b	0.043 ± 0.008ab,a,b	7.25 ± 0.60a,a,a	0.28 ± 0.01b,ab,b	0.49 ± 0.04a,a,a
	N	4.58 ± 0.39b,a,b	0.58 ± 0.10b,a,b	0.033 ± 0.003ab,a,b	8.15 ± 0.73a,a,b	0.25 ± 0.02b,a,b	0.48 ± 0.03ab,a,b
150 mM	NS	3.41 ± 0.06b,a,d	0.49 ± 0.02b,a,c	0.036± 0.003b,a,b	6.96 ± 0.47ab,a,ab	0.240± 0.00b,a,c	0.60 ± 0.03a,a,b
	PN	3.96 ± 0.19b,ab,b	0.56 ± 0.07ab,a,c	0.046 ± 0.008ab,a,c	7.26 ± 0.82ab,a,ab	0.29 ± 0.02b,ab,c	0.62 ± 0.04b,a,c
	PK	3.95 ± 0.12b,ab,c	0.57 ± 0.00b,a,b	0.046 ± 0.003a,a,b	6.90 ± 0.21ab,a,a	0.34 ± 0.03b,b,b	0.63 ± 0.01a,a,b
	N	4.65 ± 0.19b,b,b	0.54 ± 0.02b,a,b	0.040 ± 0.000b,a,b	8.54 ± 0.53a,a,b	0.24 ± 0.01b,a,b	0.53 ± 0.03b,a,ab
August
0 mM	NS	4.99 ± 0.21c,a,a	0.99 ± 0.08b,a,a	0.053 ± 0.006b,a,a	5.11 ± 0.54a,a,a	0.38 ± 0.01b,a,a	0.60 ± 0.05a,a,a
	PN	6.40 ± 0.15c,b,a	1.20 ± 0.08bc,a,a	0.066 ± 0.006b,a,a	5.33 ± 0.21b,a,a	0.42 ± 0.02c,a,ab	0.57 ± 0.02ab,a,a
	PK	7.96 ± 0.47ab,b,a	1.61 ± 0.06b,b,a	0.090 ± 0.005b,b,a	4.94 ± 0.28c,a,a	0.47 ± 0.03ab,a,a	0.59 ± 0.02b,a,b
	N	6.78 ± 0.28b,b,a	1.31 ± 0.04ab,b,a	0.063 ± 0.003bc,a,a	5.18 ± 0.24b,a,a	0.40 ± 0.02b,a,a	0.53 ± 0.02b,a,a
50 mM	NS	3.47 ± 0.18c,ab,b	0.53 ± 0.03c,a,b	0.020 ± 0.000b,a,b	6.57 ± 0.14ab,a,a	0.20 ± 0.01c,a,b	0.37 ± 0.01a,a,b
	PN	5.20 ± 0.31c,b,a	0.50 ± 0.05c,a,b	0.033 ± 0.003b,a,b	10.80 ± 1.75b,a,b	0.31 ± 0.03c,b,bc	0.40 ± 0.08a,ab,a
	PK	3.37 ± 0.49c,ab,b	0.34 ± 0.05c,a,b	0.020 ± 0.005b,a,b	10.05 ± 1.17a,a,b	0.19 ± 0.02c,a,b	0.41 ± 0.02a,ab,a
	N	3.31 ± 0.18c,a,b	0.44 ± 0.02b,a,b	0.030 ± 0.000b,a,b	7.43 ± 0.33a,a,b	0.19 ± 0.01b,a,b	0.57 ± 0.02b,b,a
100 mM	NS	2.91 ± 0.26c,a,cb	0.40 ± 0.07bc,a,bc	0.026 ± 0.003cd,a,b	7.64 ± 1.06a,a,a	0.18 ± 0.02b,a,b	0.58 ± 0.04b,a,a
	PN	5.13 ± 0.12ab,b,a	0.73 ± 0.04bc,b,b	0.046 ± 0.003b,a,ab	7.03 ± 0.37a,a,ab	0.50 ± 0.01a,b,a	0.55 ± 0.03b,a,a
	PK	2.98 ± 0.39c,a,b	0.50 ± 0.00bc,a,b	0.036 ± 0.006bc,a,b	5.91 ± 0.69a,a,a	0.24 ± 0.06bc,a,ab	0.63 ± 0.05b,a,b
	N	3.17 ± 0.31c,a,b	0.52 ± 0.03bc,a,b	0.026 ± 0.006bc,a,bc	6.05 ± 0.25b,a,ab	0.27 ± 0.02ab,a,b	0.57 ± 0.01b,a,a
150 mM	NS	2.08 ± 0.08c,a,c	0.33 ± 0.04b,a,c	0.013 ± 0.003b,a,b	6.43 ± 0.98ab,a,a	0.19 ± 0.02b,a,b	0.53 ± 0.06a,a,a
	PN	2.55 ± 0.90bc,a,b	0.48 ± 0.14ab,a,b	0.023 ± 0.008b,a,b	5.21 ± 0.56b,a,b	0.22 ± 0.07bc,a,c	0.57 ± 0.06ab,a,a
	PK	2.13 ± 0.71b,a,b	0.26 ± 0.07c,a,b	0.013 ± 0.003b,a,b	7.97 ± 0.45a,a,a	0.20 ± 0.08bc,a,b	0.40 ± 0.02c,a,a
	N	2.40 ± 0.25c,a,b	0.37 ± 0.01c,a,b	0.010 ± 0.000c,a,c	6.41 ± 0.39b,a,ab	0.22 ± 0.03b,a,b	0.43 ± 0.04ab,a,b
September
0 mM	NS	5.50 ± 0.27c,a,a	0.92 ± 0.04b,a,a	0.070 ± 0.005ab,a,a	5.99 ± 0.22a,a,a	0.37 ± 0.06b,a,a	0.64 ± 0.01a,a,b
	PN	4.89 ± 0.33d,a,a	0.86 ± 0.07c,a,a	0.060 ± 0.005b,a,a	5.67 ± 0.25ab,a,a	0.35 ± 0.02c,a,a	0.63 ± 0.01b,a,a
	PK	5.863 ± 0.3b,a,a	0.76 ± 0.04c,a,a	0.050 ± 0.005a,a,a	7.69 ± 0.61a,b,a	0.45 ± 0.03a,a,a	0.50 ± 0.04b,b,a
	N	4.57 ± 0.15c,a,a	0.78 ± 0.00c,a,a	0.050 ±0.000c,a,a	5.85 ± 0.20b,a,ab	0.39 ± 0.01b,a,a	0.61 ± 0.01c,c,a
50 mM	NS	2.18 ± 0.04d,ab,b	0.34 ± 0.01d,a,b	0.010 ± 0.000b,a,b	6.30 ± 0.14b,a,a	0.14 ± 0.01d,a,b	0.47 ± 0.01bc,a,a
	PN	1.76 ± 0.03d,b,b	0.30 ± 0.02c,a,b	0.010 ± 0.000c,a,b	5.88 ± 0.51a,a,a	0.10 ± 0.00d,a,b	0.48 ± 0.04a,a,b
	PK	3.05 ± 0.37c,a,b	0.53 ± 0.06c,b,ab	0.023 ± 0.003b,bc,b	5.87 ± 1.10b,a,a	0.19 ± 0.02c,b,b	0.49 ± 0.08a,a,a
	N	2.26 ± 0.12c,ab,b	0.39 ± 0.03b,ab,b	0.016 ± 0.003b,ac,b	5.75 ± 0.29b,a,ab	0.15 ± 0.01b,ab,b	0.50 ± 0.01ab,a,b
100 mM	NS	1.95 ± 0.29c,a,b	0.30 ± 0.02c,a,b	0.010 ± 0.000d,a,b	6.37 ± 0.42a,bc,a	0.13 ± 0.03b,a,b	0.44 ± 0.03a,a,a
	PN	1.62 ± 0.04c,a,b	0.34 ± 0.01c,a,b	0.010 ± 0.000c,a,b	4.74 ± 0.18b,a,a	0.10 ± 0.00b,a,b	0.55 ± 0.01b,b,ab
	PK	1.80 ± 0.04c,a,b	0.31 ± 0.01c,a,b	0.010 ± 0.000c,a,b	5.76 ± 0.19a,ab,a	0.10 ± 0.00c,a,b	0.47 ± 0.01a,a,a
	N	1.85 ± 0.08c,a,b	0.26 ± 0.00c,a,c	0.010 ± 0.000c,a,b	7.01 ± 0.15ab,c,b	0.11 ± 0.00c,a,c	0.34 ± 0.01c,c,c
150 mM	NS	1.50 ± 0.12c,a,b	0.40 ± 0.09b,a,b	0.020 ± 0.005b,a,b	3.99 ± 0.61b,a,b	0.09 ± 0.01c,a,b	0.65 ± 0.07a,a,b
	PN	1.23 ± 0.17c,a,b	0.21 ± 0.03b,a,b	0.010 ± 0.000b,a,b	5.82 ± 0.09b,b,a	0.08 ± 0.02c,a,b	0.52 ± 0.02ab,a,b
	PK	2.00 ± 0.51b,a,b	0.38 ± 0.09bc,a,b	0.016 ± 0.006b,a,b	5.15 ± 0.04c,ab,a	0.13 ± 0.02c,a,b	0.60 ± 0.00ab,a,a
	N	1.24 ± 0.11d,a,c	0.24 ± 0.02c,a,c	0.010 ± 0.000c,a,b	5.17 ± 0.42b,ab,a	0.09 ± 0.01c,a,c	0.63 ± 0.01b,a,a

⁽¹⁾ The different letters indicate a significant (p < 0.05) difference. The first letter demonstrates differences between months for the same exogenous treatment and level of salinity stress, the second demonstrates differences among exogenous treatments for the same month and level of salinity stress, and the third demonstrates differences for the same exogenous treatment between salinity stress levels for the same monthly period. Data are presented as the mean ± SE of five replicates. ⁽²⁾ Treat: treatment type; NS: non-supplemented olives; PN: L-methionine-supplemented; PK: choline-Cl-supplemented; N: L-proline betaine-supplemented.

).

Saline stress suppressed photosynthetic rates with time and dose. Overall recorded losses for unsupplemented plantlets under 50–150 mM soil salt stress were observed for June at 17.38–28.81%, July at 14.91–52.90%, August at 30.46–58.31%, and September at 60.36–72.72%. Additionally, 50 mM NaCl soil-stressed and amino acid-treated plantlets showed better photosynthetic rates in June, a phenomenon which remained consistent only for L-methionine application. L-methionine also positively contributed to 100 mM saline stress throughout the experimental period. The highest levels of stress (150 mM NaCl) did not provide differences between treated and non-treated olive var. Koroneiki plantlets for the same monthly period.

Analyzing data among these two varieties, ‘Koroneiki’ and ‘Kalamon’ appeared to have different trends in photosynthetic rates from June to July; ‘Koroneiki’ demonstrated values peaked in June, whereas ‘Kalamon’ showed a steady decline pattern from June to September. During the first three months, ‘Kalamon’ exhibited superior photosynthetic rates (12.32%, 50.88%, and 36.27%, respectively) in comparison to ‘Koroneiki’, which appeared with higher values in September (48.36%). Soil saline stress altered the unchallenged profiles of both olive varieties; all photosynthetic rates in June for ‘Koroneiki’ plantlet lines were higher than those of ‘Kalamon’, independent of foliar treatment and applied levels of NaCl. In July, L-methionine, choline, and L-proline betaine applications had a better effect on olive var. Kalamon plantlets under unchallenged conditions. For the same period, L-methionine application on stressed plantlets (50 mM NaCl) showed higher values in the ‘Kalamon’ variety. In all other cases, comparisons between the two varieties for the same monthly period, treatments, and levels of salt stress (50–150 mM NaCl) demonstrated non-significant differences.

3.3. Leaf Transpiration

Leaf transpiration in ‘Kalamon’ variety olives was stable until August and declined thereafter. L-methionine and L-proline betaine supplementation reduced transpiration during the first 30 days of the experimental period; however, values of the parameter reinstated close to those of non-supplemented plantlets during the coming months (July–August). Saline stress reduced transpiration in time [minimum decline rates per month: June: 42.47%; July: 45.66%; August: 58.95%; and September: 78.61%] and in a dose-dependent manner [minimum decline rates per level of saline stress: 50 mM NaCl: 42.77%; 100 mM NaCl: 55.49%; 150 mM NaCl: 65.89%]. Application of choline-Cl resulted in higher transpiration rates in June (150 mM NaCl, 35.16%), July (100 mM NaCl, 47.00%), and August (150 mM NaCl, 57.14%). Foliar administration of L-methionine also increased transpiration rates in July (100 mM NaCl, 49.18%), followed by L-proline betaine in August (150 mM NaCl, 47.50%) (Table 1).

The ‘Koroneiki’ variety olives followed a similar reduction data trend over time; amino acid application on saline unchallenged plantlets did not increase leaf transpiration rates, except for choline-Cl (38.5%) and L-proline betaine (24.42%) in August. During the first experimental month (June), the transpiration rates declined by more than 33% when var. Koroneiki plantlets were exposed to 100–150 mM NaCl saline stress. Choline-Cl application found to support plantlets with higher transpiration rates in comparison to non-supplemented ones at 50 mM NaCl (September, 35.84%). Similarly, L-methionine also induced high rates at 100 mM NaCl soil presence, but at a different time period (July, 38.04%). All other treatments in both varieties did not produce significant salinity alleviation effects on transpiration rates (Table 2).

Profile comparison of the two varieties suggested no major differences in transpiration rates per time under saline, unchallenged, and saline-challenged conditions. Choline-Cl (and in a secondary boosting role, L-methionine followed by L-proline betaine) contributed to higher transpiration rates in a conditionally saline environment; however, as the data suggested, it is a time- and dose-dependent phenomenon.

3.4. Stomatal Conductance

Leaf stomatal conductance of saline, unchallenged var. Kalamon olives remained stable in June–July and thereafter declined (53.17–77.92%) during August–September. Amino acid supplementation of salt-free plantlets did not alter stomatal conductance.

Exposure of non-supplemented plantlets to salinity stress suppressed stomatal conductance as the stress levels increased. The presence of 50 mM NaCl in soil induced sharp values that fell from June to September (61.64–75.75%). Upgrading stress levels to 100 mM NaCl, plantlet leaves further declined their stomatal functionality (11.53–39.39%) during June–August, whereas conductance values remained extremely low, forming a data plateau (0.013 mmol m^−2.s⁻¹). The highest applied stress (150 mM NaCl) revealed a major decrease in stomatal conductance (79.45–85.71%) when compared to unchallenged experimental lines (Table 1).

Choline application in August and L-proline betaine application in June increased leaf stomatal conductance above non-supplemented olives by 49.09% and 39.53%, respectively, under the same saline stress regime (50 mM NaCl). Furthermore, administration of L-methionine and choline-Cl under 100 mM NaCl exposure induced better stomatal opening functionality (42.85–47.36%) in comparison to untreated var. Kalamon olives. None of the applied amino acids offered higher stomatal conductance rates in comparison to the untreated lines exposed to severe salinity stress of 150 mM NaCl at the end of the experimental period (September); rate enhancements were provided sporadically by choline (June: 43.49%; August: 69.69%) and L-proline betaine (August only: 56.52%).

Saline-free cultivated ‘Koroneiki’ plantlets increased their stomatal conductivity rates in a single case in August using choline supplementation, where the parameter was enhanced by 41.11%. Saline soil stress inhibited stomatal functionality over time [minimum decline rates per month: June: 35.92%; July: 30.26%; August: 50.94%; and September: 71.42%] and levels of stress [decline rates per level of saline stress: 50 mM NaCl: 30.26–85.71%; 100 mM NaCl: 41.74–85.71%; 150 mM NaCl: 35.92–71.42%] (Table 2). None of the foliarly applied amino acids were capable of reinstating and ameliorating stomatal conductivity values close to those of saline-free-grown ‘Koroneiki’ olives.

‘Kalamon’ exhibited higher stomatal conductivity values from June to August; however, this pattern was reversed in the final month of the experimental period.

3.5. Water Use Efficiency

No major changes in leaf water use efficiency values were recorded during the experimental period in the saline unchallenged ‘Kalamon’ variety lines (Table 1). Amino acid application did not offer greater WUE values, with the exception of L-methionine supplementation in June (22.26%). Saline stress suppressed water use efficiency in a dose-dependent manner in all non-supplemented olives (3.80–44.17%). L-methionine application to var. Kalamon plantlets under 50 mM and 150 mM NaCl soil stress offered amelioration on WUE values (14.65% and 27.29%, respectively) when compared to non-supplemented ones in June. L-proline betaine administration on leaves raised WUE values in June (21.94%) and July (26.49%) when plantlets were exposed to the highest saline concentration (150 mM NaCl).

‘Koroneiki’ olive plantlets unchallenged with saline followed the same data pattern as the ‘Kalamon’ variety, with no major differences in time (Table 2). Foliar supplementation with amino acids under unchallenged conditions did not advance leaf water use efficiency. Saline stress did not significantly increase WUE values, except in July, when olives were exposed to 100 mM NaCl soil stress. A time-related mosaic of amino acid positive contributions to WUE values was apparent; L-proline betaine supplementation increased leaf water use efficiency during the first and last months of the experimentation period (9.12–30.57%) at 100–150 mM NaCl stress exposure. In addition, L-methionine application also contributed positively (11.16–19.70%) in June and July under mild (50 mM NaCl) and high (150 mM NaCl) stress conditions.

3.6. Leaf Quantum Yield

Leaf quantum yield declined over time during the experimental period for saline-free cultivated var. Kalamon olive plantlets; the only advancement by amino acid supplementation was recorded for L-methionine in June (20.09%). For the period from June to August, saline suppressed quantum yield in time [decline rates per month: June: 55.71–74.28%; July: 44.69–78.79%; August: 60.67–71.88%] and level of stress [decline rates per level of saline stress: 50 mM NaCl: 51.47–60.67%; 100 mM NaCl: 65.71–71.88%; 150 mM NaCl: 70.40–78.79%] (Table 1). In September, leaf quantum yields were not significantly different between saline-stressed and unchallenged plantlets. Olive foliar amino acid supplementation under 50 mM NaCl exposure boosted leaf quantum yields in June [L-methionine, choline-Cl, and L-proline betaine by 20.3%, 18.42%, and 34.87%, respectively], August [L-methionine by 42.24%], and August [choline-Cl by 43.35%]. In addition, choline-Cl application increased leaf quantum yield by 32.14% at moderate levels of stress induction (100 mM NaCl). Similarly, at 150 mM NaCl salinity stress, all applied amino acids exhibited enhanced quantum yields on var. Kalamon leaves in June (20.35–44.27%), a phenomenon which was limited in August only for choline-Cl (40.71%) and L-proline betaine (39.19%). The only positive contribution of amino acid supplementation in September was found for L-proline betaine (57.05%) under 150 mM NaCl salt stress.

Unchallenged and non-supplemented var. Koroneiki olives followed the same decline value data patterns for leaf quantum yield as seen in var. Kalamon plantlets. Amino acid supplementation did not offer greater leaf quantum yields in non-stressed experimental lines. Soil salinity negatively affected the quantum yields in leaves over time [decline rates per month: June: 42.71–53.99%; July: 30.04–51.44%; August: 47.36–51.84%; September: 62.84–76.50%] and the level of stress [decline rates per level of saline stress: 50 mM NaCl: 30.04–62.84%; 100 mM NaCl: 51.44–63.66%; 150 mM NaCl: 42.17–76.50%]. L-methionine supplementation was capable of raising leaf quantum yields in this variety under the following time periods and stress conditions [July/50 mM NaCl, 24.44%; August/50 mM NaCl, 31.33%; June/100 mM NaCl, 38.38%]. Furthermore, choline-Cl demonstrated similar positive effects, but in a single case [July/50 mM NaCl, 30.02%] (Table 2).

Saline-free ‘Kalamon’ and ‘Koroneiki’ profile comparisons resulted in no differences in leaf quantum yield values, except in July, when ‘Kalamon’ overpassed ‘Koroneiki’ performance. Foliar administration of amino acids in the two varieties resulted in different efficacy profiles.

3.7. Cin/Cout Ratio

Supplementation with amino acids did not offer better Cin/Cout values in unchallenged var. Kalamon plantlets, with the exception of foliarly administered choline-Cl in August. Saline stress did not suppress the Cin/Cout parameter; no major differences were observed among amino acid-treated experimental lines and in comparison to non-treated lines, with the exception of choline-Cl supplementation, which gave better results in August (30.84%) under 150 mM NaCl soil salt stress (Table 1).

A similar trend was also apparent in olive var. Koroneiki plantlets; however, differences with better Cin/Cout values did not occur at the same time, amino acid treatment type, and levels of salt stress. The application of L-proline betaine under 50 mM NaCl soil stress provided better Cin/Cout values in August (35.08%) compared with untreated plantlets (Table 2).

When comparing the two varieties, no differences were found in the Cin/Cout ratio when no saline stress occurred. Under mild saline stress conditions (50 mM NaCl) in August, ‘Koroneiki’ showed better Cin/Cout ratios. Foliar administration of choline-Cl in July and August under 100 mM and 150 mM NaCl soil stress induced Cin/Cout value inversion phenomena [(100 mM NaCl/July: ‘Kalamon’ 0.613 ± 0.008; ‘Koroneiki’ 0.486 ± 0.044/August: ‘Kalamon’ 0.586 ± 0.067; ‘Koroneiki’ 0.626 ± 0.047) (150 mM NaCl/July: ‘Kalamon’ 0.386 ± 0.023; ‘Koroneiki’ 0.626 ± 0.006/August: ‘Kalamon’ 0.616 ± 0.026; ‘Koroneiki’ 0.403 ± 0.016)] suggesting a differential time window for amino acid-related effects on plantlets.

3.8. Total Chlorophyll Content

From June to July, non-stressed, non-supplemented olive var. Kalamon plantlets decreased their total chlorophyll content by 84% which was restored to initial levels in August and September (Figure 9). All amino acid treatments on saline unchallenged plantlets resulted in a lower to equal chlorophyll content. Overall, the presence of salinity stress suppressed the total chlorophyll content in non-supplemented experimental lines, especially at higher (150 mM) NaCl concentrations. Without any supplementation, saline-stressed ‘Kalamon’ plantlets had lower total chlorophyll content throughout the experimental period. As salt stress increased, seedlings declined in their capacity to retain total chlorophyll content. Application of 50 mM NaCl stress reduced the total chlorophyll content in the first 30 days by 19.61%; however, the plants reinstated the content towards the non-challenged status 60–90 days later (August–September). Plantlet observations under 100 mM NaCl soil salt stress conditions were similar to 50 mM NaCl ones, with the exception of July, when plants offered the most resilient mean value in total chlorophyll content (17.68 SPAD value). The highest applied saline stress (150 mM NaCl) restricted total chlorophyll content throughout the experimental period. Application of 150 mM saline stress suppressed initially total chlorophyll content by 36.3% in June, with further expansion of the phenomenon towards the end of the experimental period (losses reached 79.54% by September).

Supplementation of soil-stressed (50 mM NaCl) plantlets with amino acids did not result in a higher total chlorophyll content than that of non-supplemented olives under the same levels of stress. In July, choline-Cl-supplemented ‘Kalamon’ olives offered 1.8–2.5 times greater total chlorophyll content moving chronologically towards August than non-supplemented and saline unchallenged plantlet lines. L-proline betaine application restored the plant chlorophyll content to the levels of saline-unchallenged var. Kalamon’s olives. All exogenous treatments in August offered equal or lower values than unsupplemented stressed seedlings with L-proline betaine to report content losses (38.13%). Amino acid supplementation was not capable of alleviating positive differences in total chlorophyll content at the end of four months of experimentation (September); however, the values were significantly close to the non-supplemented salt-stressed control line.

Exogenous application of L-methionine and choline did not show any positive effect on plantlets treated with 100 mM NaCl within the first month (June) of experimentation. In contrast, L-proline betaine application resulted in a lower total chlorophyll content than the non-supplemented control line. For the rest of the experimental period, no differences were observed between supplemented and non-supplemented seedlings under the same saline soil conditions, with two exceptions regarding lower content (a) in L-methionine-treated plantlets in July and (b) in L-proline betaine-treated plantlets in August. At the end of the experimental period in September, olives had similar chlorophyll content, independent of their treatment, for the same levels of saline stress (100 mM NaCl).

For all experimental lines of ‘Koroneiki’ olives, the total chlorophyll content was high in June, followed by a sharp drop in July (72.98–82.55%), with partial recovery attempts in August willing to retain the parameter stable plateau in September (Figure 10). In June, non-stressed salt experimental lines treated with formulated choline-Cl advanced their chlorophyll content by 31.01% in comparison to non-treated plantlets. L-methionine- and L-proline betaine-supplemented seedlings demonstrated higher chlorophyll content by 6.50–8.86% compared to non-treated lines. No significant differences in chlorophyll content were observed among the treatments in July, August, and September. In contrast, when comparing types of supplementations in successive months, mean values of total chlorophyll content from July to August were increased by 2.4 to 3.1 times for all plantlets independently of their supplementation regime, with maximum performance recorded in L-proline betaine-supplemented lines. Thereafter, in September, all plantlets retained their total chlorophyll content, with the exception of non-supplemented plants, where a 24.67% decline occurred.

For the same monthly period, non-supplemented seedlings did not differ in their chlorophyll content upon salt stress increase during the first three months of the experiment. From June to September, the high/low pattern of value changes in the total chlorophyll content was retained independently of saline stress levels. In September, a decline in chlorophyll content was apparent when salt stress was applied (36% and 43% losses for 50 and 100 mM NaCl), which tended to reach a non-significant plateau (100–150 mM NaCl).

L-methionine-supplemented plantlets in June retained their chlorophyll content up to 100 mM NaCl saline stress; thereafter, the value drop was apparent. In June and September, all olive var. Koroneiki plantlet lines treated with L-methionine had greater total chlorophyll content than the non-supplemented lines for the same levels of soil salt stress. In July and August, the seedlings followed data trends similar to those of the non-supplemented seedlings upon NaCl exposure.

Under non-saline conditions, exogenous application of choline-Cl provided an initial 30-day highest peak (June) in chlorophyll content among all experimental lines. However, seedlings lowered their performance by approximately 31% when salt stress was applied (50 mM and 100 mM NaCl) and declined further (150 mM NaCl) with 61.07% content losses when compared to choline-supplemented, saline-free plantlets. In July and August, choline-Cl-treated plantlets exhibited similar data patterns and values as non-supplemented and L-methionine- and L-proline betaine-supplemented var. Koroneiki olives. In July, moderately to severely saline-stressed plantlets (100 mM-150 mM NaCl) sharply reduced their total chlorophyll content up to 51.36%. September values revealed a resilience trend of choline-Cl-treated plantlets over total chlorophyll content in comparison to exogenously non-supplemented experimental lines; however, seedling performance was lower than that of methionine-treated plants.

L-proline betaine treatment of olive var. Koroneiki appeared to have a significant positive effect in the first month (June) of the experimental period compared to non-supplemented seedlings. The July data trend for L-proline betaine-supplemented seedlings demonstrated a similar pattern to the rest of the experimental lines. In contrast, in August, seedlings performed better under all saline stress levels (50–100 mM NaCl) and provided an under-stress total chlorophyll content surplus (18.97–21.03%) in comparison to non-supplemented ones.

At 50 mM salt stress conditions, L-proline betaine application provided higher values of total chlorophyll content in June and August. L-methionine supplementation gave good results in June and September, revealing a more resilient, long-term strategy to sustain the total chlorophyll content among all the experimental lines. Seedlings treated with choline-Cl did not perform significantly better than non-supplemented plantlets at specific levels of salt stress.

No differences were apparent among the different treatments when seedlings were exposed to 100 mM NaCl soil stress; the only exception was the methionine-supplemented experimental line, where the September plantlets provided the highest average total chlorophyll content values.

Soil salt exposure of olive var. Koroneiki at 150 mM NaCl provided a value range for total chlorophyll content throughout the experimental period without significant differences among them. Choline-Cl-supplemented seedlings provided the lowest total chlorophyll content values in June, attempting to restore them closely to other experimental lines in August and September.

Olive var. Kalamon under unsupplemented, saline-free conditions demonstrated greater total chlorophyll content than var. Koroneiki. Furthermore, ‘Kalamon’ sustained its total chlorophyll content, particularly in August, with a minor decline in September. In June, ‘Koroneiki’ had 27.75% less content than ‘Kalamon’; amplified differences between these two cultivars were more apparent in August and September (47.55% and 51.90%, respectively). Due to stressful climate conditions in July, plantlets of the two varieties narrowed their differences, a data pattern that was also observed under saline soil challenge conditions.

Under all levels of applied soil saline stress, unsupplemented seedlings of var. Koroneiki demonstrated lower total chlorophyll content for the months of June, August, and September, with augmented differences in trends. Saline stress at 100 mM NaCl resulted in the greatest difference in total chlorophyll content between the two varieties (57.89%) observed in September. Due to high levels of saline stress (150 mM NaCl), differences between the two varieties decreased to the point that they were not significant in the last month of the experimental period. A similar data profile, namely, without significant differences, was recorded in June, where data must be analyzed in comparison to monthly climate data in the area.

Plantlet supplementation of L-methionine in the unchallenged status minimized the quantitative overall superiority of the ‘Kalamon’ variety in total chlorophyll content. Saline-stressed (50 mM and 100 mM NaCl) young olive trees supplemented with L-methionine did not differ in their content when compared for the same time period. Under high saline stress conditions (150 mM), olive trees var. Kalamon demonstrated an increase in total chlorophyll content, especially in August and September, with recorded value differences of 47.85% and 40.53%, respectively.

Choline supplementation on olive trees var. Koroneiki turned out to be unable to bridge the gap with var. Kalamon’s olive performance in the last two months of experimentation (August, September) was independent of their saline challenge level (0–150 mM NaCl). Both saline-free olive varieties, supplemented with choline-Cl, appeared to have common data trends in total chlorophyll content for the first 90 days; thereafter, ‘Kalamon’ olives demonstrated their content superiority by 58.14% (August) and 54.55% (September) when compared to ‘Koroneiki’. Challenging choline-supplemented olives with 50 mM NaCl soil stress suppressed cultivar differences in total chlorophyll content, with the exception of August, when var. Koroneiki’s content was found to be lower by 60.42%. Increasing soil saline stress (100 mM NaCl) caused August content differences to remain at similar levels (58.94%), and the lagging content pattern was also quite apparent in September (37.54%). The highest saline stress conditions (150 mM NaCl) significantly lowered total chlorophyll content in a proportional manner in the two choline-supplemented olive varieties; however, at the end of the experimental period (September), differences ceased to exist.

Non-stressed olives supplemented with L-proline betaine boosted, phasing out the differences in total chlorophyll content for the May–August period. In September, olives returned to their control standards, emerging as olive var. Kalamon that is richer in total chlorophyll content. All ‘Kalamon’ saline-treated (50–150 mM NaCl) and L-proline betaine-supplemented olives revealed equal or higher ability to sustain total chlorophyll content in comparison to ‘Koroneiki’ ones (May–September).

3.9. Leaf Anthocyanin Content

For soil salt-free-grown olive var. Kalamon plantlets, the total anthocyanin concentration was high in June. Thereafter, a sharp decline (60.42%) and slow recovery of the content occurred until the end of the experiment (Figure 11). Foliar application of amino acids under non-saline stress conditions initially did not quantitatively alter the leaf content. However, in July and August, L-methionine suppressed anthocyanin production by 33.50% and 37.32%, respectively. Post-June administration of L-proline betaine induced a series of highs and lows of the content that did not follow the untreated plantlet profile. No stable high or low trend of anthocyanins was found for the entire experimental period due to amino acid supplementation.

The highest applied saline stress (150 mM NaCl) during June–August resulted in higher anthocyanin content [June: 4.69; July: 25.70%; August: 24.98%] in comparison to non-stressed trees. No differences were recorded in the anthocyanin content in September among the stressed and non-stressed ‘Kalamon’ variety olives. Foliar application of amino acids on stressed plantlets was noted to have sporadically positive contributions towards the concentration increase in anthocyanins (choline-CI in August at a 50 mM NaCl level of stress; L-methionine and choline-CI in June at a 100 mM NaCl level of stress; choline-Cl in September at a 150 mM NaCl level of stress. In contrast, low anthocyanin content in the presence of plantlet supplementation was recorded in June for 50 mM NaCl stress under L-proline betaine supplementation (58.61%), in September for 50 mM NaCl, and for all types of amino acids (22.12–43.59%). The highest salt soil presence (150 mM NaCl) coupled with amino acid supplementation exhibited low anthocyanin content in July by choline-Cl (24.57%) and L-proline betaine (42.07%) and in August by L-methionine (39.52%) and L-proline betaine (62.32%) when compared to unsupplemented plantlets.

Olive var. Koroneiki without exposure to saline stress demonstrated high anthocyanin content in June, which thereafter sharply reduced (51.45%) without recovery (Figure 12). L-methionine supplementation increased the content in August (33.24%). No decline in anthocyanin content was observed due to foliar amino acid supplementation in saline-unchallenged plantlets.

Salinity increased anthocyanin content from the beginning of this study (June), even at the lowest applied NaCl concentration, a data trend that continued in July. Higher levels of soil salt stress (e.g., 100–150 mM NaCl) in a stable mode resulted in higher anthocyanin content throughout the experimental period.

Amino acid supplementation in the presence of 100 mM NaCl soil did not affect the total anthocyanin content, with the exception of L-proline betaine application in June, which was lower (26.35%) than that in non-treated plantlets. Furthermore, the same type of supplementation at 150 mM NaCl salt stress did not affect anthocyanin content, except for L-proline betaine supplementation in September, where values were found to be higher in comparison to non-treated experimental lines.

‘Kalamon’ olives produced more anthocyanins in leaves than ‘Koroneiki’ under zero saline conditions, with the exception of July, when the two varieties shared similar data values. The applied stress of 50 mM NaCl kept the same comparable profile values trend (e.g., greater anthocyanin content in ‘Kalamon’ leaves) with the expansion of one month more of non-significant content differences (July–August). Increasing the level of stress (100 mM NaCl) reversed the recorded anthocyanins’ zero saline-challenged content profile, with higher anthocyanins observed in the ‘Koroneiki’ variety in August and no differences among varieties for the rest of the experimental period. The highest levels of salt soil presence (150 mM NaCl) offered a ‘Kalamon’ variety comparative content advantage for June, which was eliminated in the months that followed (July–September).

3.10. Total Phenolics

Technically, the application of amino acids to saline-unchallenged ‘Koroneiki’ variety plantlets did not alter the content of total phenolics (Figure 13). The effect of salinity on non-supplemented olives slightly decreased the phenolic content. L-methionine appeared to increase total phenolics under 50 mM and 150 mM NaCl stress, followed by choline, which raised the relative content only at 150 mM NaCl soil presence. L-proline betaine steadily promoted an increase in phenolic content at all levels of saline stress.

Soil saline-free-grown var. Kalamon olives did not change their total phenolic content by foliar administration of amino acids. However, when exposed to saline (150 mM NaCl) without any supplementation, leaf phenolic content was reduced (Figure 13). All amino acids applied at 150 mM NaCl stressed grown olives reinstated and exceeded (13.41–14.47%) the total phenolic content in leaves. Although amino acid administration on ‘Kalamon’ plantlets increased phenolics in the presence of 150 mM NaCl soil, no differences were recorded in lower levels of stress (50–100 mM NaCl), with the exception of L-methionine, where the content in leaves declined (4.56%).

3.11. Antioxidant Activity (FRAP)

A mosaic of responses was recorded when var. Kalamon plantlets were exposed to an exogenous amino acid delivery scheme under zero challenge of salinity stress. Application of choline-Cl and L-proline betaine reduced leaf FRAP values, whereas L-methionine slightly increased them above those recorded in the non-supplemented experimental lines. Saline stress declined FRAP values in a dose-dependent manner. L-methionine and choline-Cl boosted leaf FRAP values when olive var. Kalamon’s plantlets were exposed to 50 mM and 100 mM NaCl soil stress. L-proline betaine reduced FRAP values in leaves at all salinity-stressed plantlets. All applicable materials reduced FRAP values at the highest applied salinity stress level (150 mM NaCl) (Figure 14).

Without saline stress, var. Koroneiki olives reduced their FRAP values under all amino acid supplementation (6.32–35.66%) in the following sequence from high to low: L-methionine > L-proline betaine > choline-Cl. The presence of saline stress increased FRAP values at middle and high levels of saline stress exposure (100–150 mM NaCl). All foliarly administered amino acids increased leaf FRAP values when plantlets were exposed to all saline stress levels, with the exception of L-methionine at 100 mM NaCl in terms of presence in the soil. The highest induced FRAP values were recorded for L-methionine at 150 mM NaCl (17.02%), choline-Cl at 50 mM NaCl (45.02%), and L-proline betaine at 50 mM NaCl (31.16%). The efficiency of choline-Cl and L-proline betaine towards higher FRAP values was reduced as plantlets reached higher levels of saline stress (Figure 14).

Comparison between the two varieties reveals higher antioxidant activity for the olive var. Kalamon under zero and 50 mM NaCl saline stress, whereas var. Koroneiki is found to be more active when exposed to middle and high levels of saline conditions.

4. Discussion

Historically, human interventions to alleviate salinity stress effects in olive groves have always been multi-parametric and deeply interdisciplinary: from water management practices [101] to soil amendments [102,103], precision agriculture [104,105], soil sensing [106,107], and in silico search for potential solutions to harsh salinity stress scenarios [108,109]. In this work, we used different types of formulated amino acids to induce resilient phenotypic plasticity in two olive varieties for undisruptive oil and raw fruit production under saline stress conditions. Under non-saline challenge conditions, var. Kalamon had declined growth rates in August; this phenomenon is due to the beginning of a new phase of the vegetative productive cycle (late summer–autumn), as has been recorded for other olive varieties [110]. Supplementation with the tested amino acids was not capable of boosting growth rates for the tested period because of the biosynthetic arrangements of both tree varieties, which are apparent even in the plantlet stage, where fruit growth and development is a forthcoming primary physiological goal [111]. Saline conditions revealed vegetal growth resilience of ‘Kalamon’ and ‘Koroneiki’ varieties, as has been documented by other research groups [25,112].

Salinity and amino acid supplementation did not have an effect on ‘Koroneiki’ concerning the number of laterals; this advanced phenotypic profile serves a dual role: (a) saline stress growth resilience, (b) tolerance to exogenous amino acid applications since there are cases like in Pyrus spinosa where administration of amino acids like L-methionine reduced the number of lateral shoots, and (c) tissue spatial signaling differentiation was unaffected by the stress factor [113,114]. A low number of lateral shoots was observed in var. Kalamon plantlets grown in saline-free conditions when choline chloride was applied, which may reflect the underlying stress of choline incorporation in tree structural and biochemical processes (membrane phospholipids, enzymes, and specific transporters), where signaling or functional roles promote vegetative growth, as documented in plant model organisms [115,116,117]. Furthermore, in excessive spatial–temporal exposure to Cl ions, plants can inhibit growth [118,119].

The decline of ‘Koroneiki’ in terms of lateral shoot length above 50% under 150 mM NaCl soil stress in the absence of amino acid supplementation was due to negative physiological regulation and cellular stress signaling in apical meristems [120]. Transcriptional factors in olive trees belonging to the WRKY and bZIP families were found to be related to shoot growth inhibition when exposed to salinity stress [121,122]. L-methionine and L-proline betaine counteracted the advancement of lateral shoots, as was also seen in other tree species, which may be due to different cellular metabolism prioritization when these amino acids are administered under salt-free soil conditions [27,123]. ‘Kalamon’ variety exhibited a resilient pattern of lower phenotypic plasticity for shoots lateral length compared to ‘Kalamon’ for the same studied factor, independently of amino acid supplementation; this may be due to hormonal balancing mechanisms when amino acids are available in diverse concentrations [124]. Soil salinity stress on var. Koroneiki decreases lateral length values, as described in many other olive varieties [23].

Most of the amino acids appeared to have no to zero balance effect on the ‘Kalamon’ variety on photosynthetic rates with the decline exemptions of L-proline betaine (June) and choline chloride (August) applications reaching data trends of non-supplemented experimental lines in September; this plasticity of responses may be due to high seasonal thermal stress and/or potential physicochemical limitations in exogenous amino acid use. Betaine enhanced photosynthetic rates when applied to ‘Chondrolia Chalkidikis’ olive trees to alleviate heat stress under drought conditions [125]; the decline in photosynthetic rates under the choline chloride supplementation scheme does not share the same trend as choline analog molecules in plant tissue culture systems [126].

Exogenous administration of L-proline betaine ameliorated photosynthetic rates in both varieties under saline exposure; similar results were reported for the ‘Chemlali’ olive variety, where only proline was found to have a dominant supportive role against this type of stress [127,128]. Positive contribution of choline-Cl to the ‘Kalamon’ variety under the same saline stress levels of 150 mM NaCl was also reported for the cluster bean Cyamopsis tetragonoloba [129].

In ‘Koreiniki’, amino acid supplementation raised stressed photosynthetic rates; however, no chronological synchronization effect was apparent among amino acid treatments. In August, ‘Koroneiki’ plantlets were resilient under saline stress with all types of exogenous administration. Differences in photosynthetic rate performance among olive cultivars under saline stress for extended periods have also been reported by other research groups [130,131].

Olive leaf transpiration can be affected by thermal conditions and salinity stress [132,133]. For the ‘Kalamon’ variety, applications of choline-Cl and L-proline betaine attempted to reinstate the regular transpiration rate towards the unchallenged status. Alleviation of salinity stress effects with restored transpiration rates was reported for (a) celery, rice, and potato plants when exposed to exogenous treatment with proline [134,135,136]; (b) rice plants exposed to exogenous choline chloride [137]; and (c) cotton plants exposed to glycine betaine [138]. A similar response profile in the ‘Koroneiki’ variety suggests that transpiration rates under saline stress are regulated by a potentially unified mechanism in which osmoregulatory amino acids are able to contribute.

Decline in stomatal conductance was apparent for both varieties with saline exposure, and this phenomenon has also been documented for other olive varieties, such as ‘Zard’, ‘Abou-satl’, ‘Arbequina’, ‘Arbosana’, ‘Chryssophylli Ntopia’, and ‘Lefkolia’ [130,131,133,139]. However, it is not a universal pattern response in all olive cultivars since the varieties ‘Arvanitolia’, ‘Atsicholou’, ‘Chemlali’, ‘Chetoui’, and ‘Smertolia’ have been reported to be resilient to this parameter [130,139]. Because of the large intra-varietal genetic variation in olive varieties, it is possible that stomatal conductance data do not follow the same trends, especially for var. Koroneiki [130,140].

Leaf water use efficiency values were not boosted by amino acid application when the experimental lines remained saline-unchallenged. The parameter was suppressed by soil salinity, and restoration patterns were recorded when L-proline betaine was administered to both varieties; similar results were observed for the ‘Zard’ olive variety [133]. Furthermore, under applied soil stress, ‘Koroneiki’ plantlets were capable of increasing their leaf water use efficiency via L-methionine supplementation; however, administration of amino acids is not the only way to inhibit water use decreasing functionality, since biopriming techniques are also capable of achieving the same results in olive trees [141].

Leaf quantum yield was attempted to be restored by all types of amino acid supplementation in both olive cultivars; however, none of them were consistently supportive throughout the experimental period. Data fluctuations during the summer period for olive plantlets var. Koroneiki are also reported by other research groups [142]. Induction of higher leaf quantum yields due to exogenous proline supplementation under saline stress has been widely documented for non-tree species such as Triticum aestivum, Cakile maritima, Brassica juncea, and Aloe vera [143,144,145,146]. Furthermore, betaine treatment increased quantum leaf yields in Cucumis sativus and Zea mays [147,148].

Resilient var. Kalamon’s ratio Cin/Cout data are in accordance with other research groups, documenting as sensitive olive plantlets those from ‘Megaritiki’ and ‘Kothreiki’ varieties [149]. Even at low stress levels, olive ‘Koroneiki’ sustained its gas exchange features due to prolonged choline chloride supplementation; this varietal weakness has also been reported without exogenous supplementation [149,150]. A similar supplementation effect was recorded in rice [137].

All amino acid supplementations during the critical period of July–August attempted to reinstate the total chlorophyll content with the application of L-proline betaine to confer better, close to saline, unchallenged status results. It is important to note that the month of July was suboptimal regarding temperature and humidity for the tested olive plantlets. Exogenous administration of proline contributed similarly to the olive varieties ‘Zard’, ‘Arbequina’, and ‘Arbosana’ [151,152]. L-methionine also had positive effects on the total chlorophyll values in both varieties; however, this attribute could reach a plateau, as proven in other species, if salinity stress memory is involved [114].

Total phenolic content for both varieties slightly declined in the presence of salinity stress; this response is apparent in plant species when (a) the variety or examined intra-varietal genetic profile is susceptible to this type of stress [140,153,154] and (b) NaCl is administered to plants via incorporation of the latter into liquid nutritive medium [155]. For tree species, total phenolic content decreased in the leaves of Prunus amygdalus Garmen GN15 and in the roots of P. amygdalus GF677 rootstocks when exposed to saline stress [156,157]. Administration of amino acids appeared to contribute to higher phenolic contents in both varieties only when the highest level of stress was applied, with exogenous application of (a) proline recorded to have a positive total phenolic induction in Aloe vera, Apium graveliens [134,145]; (b) betaine observed to enhance phenolic content in saline exposed Carthamus tinctorius [158]; and (c) methionine to increase phenolic content in Zea mays and Spinacia oleracea [41,159]. Halophylic coastline environments for tree species do not offer great opportunities for phenolic enhancement after exogenous L-methionine application due to long-term adaptation mechanisms [114].

Anthocyanins alleviate the effects of saline exposure by adjusting osmotic balance, ion homeostasis, and antioxidant defense; both olive varieties increased their anthocyanin content upon 100–150 mM NaCl exposure [160]. Apple trees utilize anthocyanins for antioxidant defense under saline conditions. Genetically modified overexpression of MdZAT5, a C2H2-type zinc finger protein in apple Calli, confers resilience in saline environments [161]. Supplementation of stressed olive plantlets with amino acids did not alter anthocyanin content, suggesting a more complex and specific alleviation of stress response where epigenetic effects and hormonal signaling could potentially be involved [160]. Increased FRAP values when olive trees var. Koroneiki exposed to salinity stress are in accordance with other research groups [162]; however, var. Kalamon exhibited a different antioxidant profile than ‘Koroneiki’ experimental lines.

Throughout this work, none of the supplements’ amino acids offered a continuously stable effect. This may be due to (a) physiological, unchallenged alterations of olive tree nutritional needs from June to September; (b) stress physiology in the presence of extreme heat weather; (c) qualitative or quantitative drift and/or delays of molecular sensing from the outer environment; (d) naturally occurring microbial flora on plantlets that may exploit faster amino acid supplementation in comparison to olive tissues; (e) speed and effectiveness of systemic/local transport mechanisms for the administered amino acids [163,164,165]; (f) delays in induced production of osmoregulatory molecules [166,167,168]; and (g) differential uptake and distribution of amino acids [169,170]. This amino acid contribution variability among different periods and parameters is not statistically dependent because of the non-unified basis of the phenomenon’s expression.

Furthermore, methionine in the presence of ATP and relative synthase enzyme support is capable of producing S-adenosylmethionine (SAM), a molecule involved in the synthesis of polyamines and methylation of physiologically important molecules such as hormones, neurotransmitters, lipids, proteins, and nucleic acids [171,172]. Increasing rates of endogenous SAM content due to saline stress appeared to be a varietal-dependent phenomenon, reflecting the personalized attributes of the plant genotype [173]. SAM-induced metabolic regulation of DNA and histone methylation significantly alters metabolic processes and cellular signaling, which (a) are not known yet in full extent and (b) take place on an evolutionary basis jointly with the levels of stress factor exposure [174,175]. Similarly, proline has been documented as an inducer of epigenetic effects [176] and a biosynthetically induced end product of epigenetic mechanisms that alleviates salinity stress [177,178]. As a physiological index, maintaining low proline levels is proof of alleviated post-salinity stress conditions, a phenomenon that can also be regulated by epigenetic mechanisms [176,179]. In the case of betaine/glycine betaine, epigenetic phenomena can affect endogenous glycine betaine production in Arabidopsis thaliana when exposed to saline environments [180]; however, halophilic methanogen glycine–betaine gene expression in A. thaliana was sufficient and stable under the same type of stress as incorporated to the plant genome of the model organism, without reporting epigenetic phenomena, possibly due to different induction mechanisms of expression [181]. Findings of betaine epigenetic activity in other model organisms and humans [182,183], jointly with existing plant-related tools and protocols, will reveal the exact mechanism of betaine contribution [184,185,186].

Choline chloride has been observed to induce epigenetic effects in animals with neuroprotective and postnatal body size effects; however, available information on plant organisms is still limited [187,188]. Comparison of choline effect to betaine effect for alleviating salinity stress effects may be confusing due to (a) microbial choline oxidation to glycine betaine, (b) plant-based oxidation to glycine betaine, and (c) third-party mechanisms like limitation of glycine betaine synthesis due to choline import into chloroplasts [189,190].

Herein, supplementation of amino acids through foliar administration on olive plantlets is a common agronomic practice [127]; microorganisms primarily on the phyllosphere and secondarily on the soil are also exposed to this content [191,192]. In ‘Koroneiki’ olive trees, bacteria from the genera of Pseudomonas, Acinetobacter, Staphylococcus, Rhodococcus, and Micrococcus are present in the phyllosphere and orchard soil [192]. Some members of these genera are capable of expressing halotolerant behavior, uptake, transport, and virulence modification and their levels of interaction with plant immunity when exposed to methionine [193,194,195], proline [194,196,197,198,199,200,201], betaine [202,203,204,205,206], and choline [206,207]. An in-depth investigation of the role of exogenous amino acid administration on plant species under saline stress and the orchard microbiome is needed to describe the exact ecosystemic fate of amino acids and understand their net contribution to this multi-partite interaction. It is worth mentioning that amino acids like those used in this work (e.g., proline) are exudated by tree roots in case of abiotic stress affecting interactions with soil microflora, notifying biotic soil agents of their physiological stress status [208,209,210].

Unfortunately, there was no amino acid supplementation among the tested ones to ameliorate all the declined physiological and biochemical parameters simultaneously; this led to the creation of varietal, time-lapse mosaic sets of responses with potentially underlying epigenetic phenomena and microbial interference for the open-field-grown olives. To overcome this problem and achieve a more resilient set of responses for the entire experimental period, a selective dose, type (foliar/soil), and time application plan is needed, considering (a) the varietal functionality of the olive genome and (b) the collective responses of the orchard microbiome under saline stress conditions. Alleviation of salinity stress must be highly prioritized on the basis of the importance of the study parameter; salinity stress is a multi-parametric damaging phenomenon on olive plantlet physiology, and priority must be given to restore the most important mechanisms, taking into account the application timing and the intensity of salt-stress caused damage.

5. Conclusions

Amino acid supplementation for both olive varieties did not positively alter their lateral length and number, a phenomenon that occurred independently of saline exposure levels. Photosynthetic rates declined compared with the unchallenged status. ‘Kalamon’ variety under low levels (50 mΜ NaCl) of salinity stress offered better photosynthetic rates under L-proline betaine and L-methionine supplementation. Only L-proline betaine and choline chloride offered mild (50 mΜ NaCl) and severe (150 mΜ NaCl) levels of saline exposure resilience in this parameter, respectively. In contrast, ‘Koroneiki’ experimental lines under the same type of stress conferred amelioration of the parameter by L-methionine only at 50–100 mM NaCl exposure. Stressed olive plantlets of both varieties achieved higher leaf transpiration rates in the following order: choline-Cl > L-methionine > L-proline betaine. Choline chloride supported stomatal conductance in stressed ‘Kalamon’ olives; this pattern was not followed by ‘Koroneiki’, where none of the amino acids induced greater values of the parameter. Water use efficiency for both varieties under stress was supplemented by amino acids; however, time-related mosaic responses suggest a hard path to lead towards a solid agronomic practice formation. Under salinity stress, patterns of quantum leaf yield increased due to exogenous treatments; however, there were differences among supplemented olive varieties. Also, the Cin/Cout ratio was not affected during the abiotic challenge. In contrast, total phenolics and anthocyanins in leaves declined when stress factors were applied; exogenous applications were not found to be efficient for all levels of saline stress. In all cases, mosaics of responses for all exogenous treatments were recorded during the experimental period (June–September), suggesting either a maximum of 1–2 months effect of amino acid supplementation on stressed olive plantlets (for the same levels of soil salinity stress) or a mosaic of responses among various salinity stress levels, which requires further in-depth mechanistic investigation.

This work focused on the responses of olive trees to L-methionine, choline chloride, and L-proline betaine foliar administration to alleviate saline stress effects. None of the applied materials on olive trees could stand alone to mitigate stress (a) among different salinity levels and (b) long-term seasonal exposure. Understanding the assimilation rates of exogenously administered amino acids in olive trees and the value of residual, nutritionally opportunistic amino acid deposits for the olive orchard microbiome could provide better perspectives for integrated strategies to mitigate salinity stress.

For some of the studied parameters, an in vitro experimentation for both varieties will be capable of producing at the micro-scale and in a short time, scheduled plans with combinedly applicable amino acid choices for desired agronomic results. Amino acids are capable of reinstating the unchallenged physiological status of olive trees; however, they do not last long and are not effective for all levels of salinity stress, a condition which reflects the norm in salinity-affected orchards.

The recorded differences between the two supplemented olive cultivars in this work highlight the need for a global, more detailed labelling in directions for use. Products with active ingredients like those tested herein must provide varietal and time-window information for application since (a) the duration of their effects is not recorded as at least seasonal, and (b) there was no agronomically scalable response upon stress exposure.