Basil as Secondary Crop in Cascade Hydroponics: Exploring Salinity Tolerance Limits in Terms of Growth, Amino Acid Profile, and Nutrient Composition

In a cascade hydroponic system, the used nutrient solution drained from a primary crop is directed to a secondary crop, enhancing resource-use efficiency while minimizing waste. Nevertheless, the inevitably increased EC of the drainage solution requires salinity-tolerant crops. The present study explored the salinity-tolerance thresholds of basil to evaluate its potential use as a secondary crop in a cascade system. Two distinct but complemented approaches were used; the first experiment examined basil response to increased levels of salinity (5, 10 and 15 dS m−1, compared with 2 dS m−1 of control) to identify the limits, and the second experiment employed a cascade system with cucumber as a primary crop to monitor basil responses to the drainage solution of 3.2 dS m−1. Growth, ascorbate content, nutrient concentration, and total amino acid concentration and profile were determined in both experiments. Various aspects of basil growth and biochemical performance collectively indicated the 5 dS m−1 salinity level as the upper limit/threshold of tolerance to stress. Higher salinity levels considerably suppressed fresh weight production, though the total concentration of amino acids showed a sevenfold increase under 15 dS m−1 and 4.5-fold under 5 and 10 dS m−1 compared to the control. The performance of basil in the cascade system was subject to a compromise between a reduction of fresh produce and an increase of total amino acids and ascorbate content. This outcome indicated that basil performed well under the conditions and the system employed in the present study, and might be a good candidate for use as a secondary crop in cascade-hydroponics systems.


Introduction
Enhanced soil salinity is a worldwide and expanding problem posing serious threats to crop production [1]. It is an inherent problem of intensive cultivation systems and of semi-arid zones, which are characterized by the imbalance between precipitation and evapotranspiration. Nevertheless, increased salinity affects soilless cultivation systems as well-either open or closed [2]. Especially in the latter, where the nutrient solution re-circulates more than once in the crop lines, the increased salt accumulation in the root zone is inevitable. This entails risks regarding impaired plant function and performance, which negatively affect crop yield [3,4]. Additionally, in both open and closed soilless systems, the discharge of used nutrient solutions to the environment further deteriorates soil quality, causing severe environmental degradation and a waste of resources [5]. The ultimate result of such management practices is a reduced sustainability of soilless systems, although their implementation has considerable advantages in terms of crop productivity, space utilization, and nutrient-use efficiency [6,7]. Toward mitigating the environmental impacts of the discharge of waste nutrient solutions, a new concept in closed systems has

Plant Material and Experimental Design
This study was conducted in the greenhouse premises of Hochschule Geisenheim University in Geisenheim, Germany during the summer months of 2018. The mean monthly temperature during the experimental period ranged from 22.4 to 27.0 • C, and crops were grown under ambient light conditions, with 517 µmol m −2 s −1 average light intensity. Basil seeds of the Genovese (Eowyn) variety were sown in 3 L pots containing a mixture of peat and perlite (2:1). After two weeks, basil plants had reached the two true leaves stage. For the first experiment, a total of 40 pots with 25 plants each were selected. The pots were divided into four treatments with 10 replicates each, and were irrigated daily with a nutrient solution of four salinity levels; i.e., 2 dS m −1 (control), 5 dS m −1 (T5), 10 dS m −1 (T10), and 15 dS m −1 (T15). Commercial fertilizers were used to build up the selected salinity levels. Table 1 summarizes the elemental composition and concentration of each nutrient in the irrigation solution used for the four treatments. The pots were arranged according to the randomized complete block design, and frequent rotation (every 10 days) was performed to minimize the impact of the microenvironment. The experimental period lasted five weeks, during which two harvests were performed, 15 and 35 days after commencement of the salinity treatment. In the second experiment, a cascade system was established with cucumber as a primary crop grown in hydroponics, the drainage of which was driven to basil grown in pots as a secondary crop. A total of 36 cucumber plants were planted in six rows, each composed of two rock wool slabs (Grodan, Roermond, the Netherlands; length: 1 m; volume: 11.25 L) planted with three cucumber plants each. The primary crop plants were allocated to three groups, with 12 plants each. The drainage of each group was driven to a tray upon which 12 pots were placed and received this capillary irrigation, with no additional watering throughout the experimental period. Each pot contained 25 basil plants, as described above. Thus, three replicates of the drainage solution treatment were formed while the control group of basil (also 12 pots) received fresh nutrient solution, the same that was prepared for cucumber. The latter was a standard nutrient solution for cucumber grown in open hydroponic systems, with the following composition: 3.0 mM K + , 6.0 mM Ca 2+ , 2.0 mM Mg 2+ , 1.0 mM NH 4 + , 11.5 mM NO 3 − , 1.5 mM H 2 PO 4 − , 3.5 mM SO 4 2− . The electrical conductivity (EC) was set at 2.1 dS m −1 and pH 5.7. The EC range of the drainage solution that irrigated the treated basil plants was 3.2 ± 0.3 dS m −1 , and its composition was 4.1 ± 0.3 mM K + , 7.1 ± 1.0 mM Ca 2+ , 2.2 ± 0.6 mM Mg 2+ , 13.2 ± 2.8 mM N, 1.7 ± 0.3 mM P + (average ± SD from three measurements during the experimental period). The same experimental duration and harvest times as for the first experiment were applied.
The following methods and sampling protocols for the growth and biochemical parameters determination apply to both experiments described above.

Plant Growth
Plant height was measured at intermediate and final harvest. At the same time-points, projected leaf area was determined through capturing photographs from the same height above plants and subsequently using the free software ImageJ (open-source software, ImageJ.net/ver. ImageJ 1.51j) to estimate the green area of the plants. At the intermediate and final harvests, five random plants were selected from each pot. Leaves and stems were separated, the fresh weight was measured immediately, and all the samples were oven dried for four days at 55 • C and weighed for biomass assessment.

Nutrient Element Analysis
After the determination of dry weight, 0.25 g of leaf tissue from each sample was used to conduct the nutrient elemental analysis. A modified Kjeldahl extraction was used for the mineralization of all nutrients. Each leaf sample was extracted with 4.4 mL of the digestion solution, which included 1.94 mL concentrated sulfuric acid, 2.82 mg Se, 82.13 mg Li 2 SO 4 , and 1.94 mL 30% H 2 O 2 . The samples were digested for two hours at 30 • C, then left to reach room temperature, and finally diluted up to 50 mL with distilled water before proceeding to elemental analysis. The concentrations of N, P, K, Ca, Mg, Fe, Zn, Mn, and Cu were determined by ICP-OES (Spectro Arcos EOS 12 ICP-OES Spectrometer, SPECTRO Analytical Instruments GmbH, Kleve, Germany) and flow injection analysis (Foss Tecator FIAStar 5000, FOSS, Hilleroed, Denmark). The concentration of macronutrients (N, P, K, Ca, Mg) and micronutrients (Fe, Zn, Mn, Cu) are expressed in % and mg/kg of leaf dry weight, respectively.

Ascorbic Acid Content
For each treatment, 20 individual plants were used to constitute five samples. For each plant, all the leaves were removed and grounded in liquid nitrogen. The ascorbic acid content was determined according to Pegg et al. (2007) [22]. First, 100-200 mg of tissue per sample was homogenized using 5 mL of 80% ethanol. The homogenate was mixed threefold using a vortex for 5 s. The samples were submerged in an iced ultrasound bath for 15 min. After that, the samples were centrifuged at 1792× g for 15 min at 0 • C, then 1 mL of supernatant was transferred into cryogenic Eppendorf vials and stored at -80 • C. The ascorbic acid content (mg AsA/g of dry tissue) was determined based on photochemoluminescence (PCL) with the PHOTOCHEM Antioxidant Analyzer (Analytik Jena GmbH, Jena, Germany).

Amino Acids
The sample preparation followed the procedure described above for the ascorbic acid determination. Concerning the extraction, 100-200 mg of tissue per sample were homogenized using 2 mL of polyvinylpyrrolidone (PVP) 100 buffering solution. The homogenate was mixed using a vortex for 5 s. The samples were submerged in an ultrasound bath for 15 min. After that, the samples were centrifuged at 1792× g for 15 min at 4 • C. The supernatant was filtered through a 0.2 nm cellulose filter to sealable glass vials. The concentration of amino acids (mg/kg of fresh tissue) was determined using an automatic Amino Acid Analyzer (SYKAM s433, Sykam GmbH, Fürstenfeldbruck, Germany).

Statistics
Statistical analysis was performed with IBM SPSS Statistics v.26 software, using one-way ANOVA, followed by Tukey's HSD post hoc tests, and confidence intervals for p ≤ 0.05.

Exploration of Salinity-Tolerance Thresholds of Basil
In the first experiment, we evaluated the productivity as well as quality parameters of basil exposed to various levels of salt stress to explore its tolerance thresholds. Moreover, we performed an evaluation with two time-points, including an intermediate harvest before the final one to identify crucial patterns of response and the course of growth performance.
Basil's growth response to increased salinity confirmed its moderate potential to cope with this stress condition. Plant height reduction was evident at both intermediate and final harvest ( Figure 1A). In the latter, T5 slightly reduced plant height, but with statistical significance, while T10 and T15 severely affected it, resulting in a decrease of 46 and 62%, respectively, compared to the control. After only two weeks of exposure to stress, the treated groups began to differentiate from the control plants, and these differences were magnified in the final harvest. The same profile was followed by another indicative growth parameter, the projected leaf area, as shown in Figure 1B. The between-treatment differences over the course of the experiment were clearly reflected in both intermediate and final harvest values of projected leaf area, showing a significant stepwise reduction with increasing salinity levels ( Figure 1B). The time-point of 15 days seemed to be crucial for all growth responses of basil, since it marked the establishment of the first statistically significant differences compared to the control. This applied not only to height and leaf area, but also to the plant fresh weight and dry biomass production. Figure 2A presents the fresh weight as determined in the intermediate and final harvest. After 18 days of exposure, T5 caused a small but significant decrease of 18% compared to the control, and this was maximized to 47% at the final harvest. T10 and T15 considerably suppressed fresh weight production, reaching a remarkable 72% and 87% reduction of control values, respectively, at the end of the experiment. Similar severe reduction was evidenced in basil dry biomass accumulation ( Figure 2B). In the relevant literature, there are some studies on basil indicating that certain varieties are tolerant to salinity levels even higher than those examined in the present study [20]. Nevertheless, most similar works emphasize the limited potential of basil to cope with salinities higher than 5 dS m −1 , corroborating our results [23,24]. Indeed, increased salinity was found to negatively affect basil height [12] and have a detrimental effect on basil's canopy area [25,26], while Caliskan et al. (2017) [27] indicated a negative correlation between the accumulation of dry matter and increased salinity. The various aspects of basil growth performance in the present study collectively indicated the 5 dS m −1 salinity level as the upper limit/threshold of tolerance to stress, and 15 days of treatment as the critical point for the appearance of salinity symptoms on growth. It is well documented that during the early phase of salinity stress (first days), the growth reduction is ascribed to decreased leaf emergence and expansion [28,29]. The underlying mechanisms are related to osmotic stress, which affects the availability of water to the plant body with profound effects on stomatal conductance, cell cycle, and cell expansion. Apart from the rapidly occurring water stress, the evolution of oxidative stress by uncontrolled production of ROS, as well as nutrient imbalances, may account for the compromised growth under enhanced salinity [26,30]. Accordingly, the time frame of 15 days (intermediate harvest) and, moreover, 30 days (final harvest) in the present experiment may be adequate for these stresses to be developed. In an article demonstrating both the water stress imposed and the antioxidant response of salt-affected basil, Barbieri et al. (2012) [31] reported that the constitutively reduced stomatal density improved the acclimatization of the more tolerant basil variety to salinity stress, along with the efficient production of antioxidants.
The total concentration of amino acids in basil leaves showed a sevenfold increase under 15 dS m −1 and 4.5-fold under 5 and 10 dS m −1 compared to the control at the final harvest ( Figure 3). Statistically significant but smaller differences between the treatments were also recorded in the intermediate harvest. It was noteworthy that the total amino acid content of the control plants remained virtually unchanged between the intermediate and final harvests, while saline treatments induced a three-to fourfold increase. An accumulation of free amino acids has been usually reported in various plants exposed to abiotic stress [32,33]. Neto et al. (2019) [21] measured the total content of amino acids of two basil varieties grown under 80 mM NaCl and reported a marginal increase in both leaves and roots. There is a tight relationship between amino acid metabolism and plant response to stress, due to the multiple roles of certain amino acids in stress mitigation; i.e., osmoprotectants, ROS scavengers, N source and storage, and as alternative substrates for mitochondrial respiration [32][33][34]. Whether from a direct salinityinduced effect or basil's response in the adaptation process, the increase of total amino acid content indicated metabolic adjustments and was particularly ascribed to specific compounds. To the best of our knowledge, this is the first report of salinity effects on the detailed amino acid profile of basil. Notably, it was obvious in the individual amino acid concentrations ( Table 2) that glutamine and arginine showed a significant increase at both intermediate and final harvests in all salinity levels. At the final harvest, the asparagine was also responsive to the imposed stress in a salinity level-dependent manner. In fact, the above-mentioned amino acids presented an eight-to 12-fold increase in T15 compared to the control, substantially contributing to the enhanced levels of total amino acid content shown in Figure 3. The results presented are in accordance with other authors, who suggested that amino acids such as asparagine, arginine, and glutamine, as well as proline, function as compatible solutes combating osmotic stress within plant cells [32,33]. Asparagine accumulation may also play a role in nitrogen remobilization and ammonia detoxification during abiotic stress [35], while the role of arginine as precursor of the stress-induced polyamines is well documented [32].       such as asparagine, arginine, and glutamine, as well as proline, function as compatib solutes combating osmotic stress within plant cells [32,33]. Asparagine accumulation ma also play a role in nitrogen remobilization and ammonia detoxification during abiot stress [35], while the role of arginine as precursor of the stress-induced polyamines is we documented [32].  Ascorbic acid (AsA) is directly involved in salinity stress protection, and particularly in basil, it has been identified as a good indicator of the total antioxidant capacity [36]. The AsA content of T15 plants was severely suppressed at the final harvest, although at the intermediate harvest showed a statistically significant increase compared to all other treatments ( Figure 4). Only T5 plants displayed similar AsA concentration with the control group. High levels of AsA effectively maintain low levels of H 2 O 2 , which may prevent the H 2 O 2 -mediated stress responses and can therefore contribute to overcome saline stress [31]. The enhanced AsA content at the intermediate harvest in the T15 group may reflect this process. Nevertheless, the decreased levels of AsA after prolonged salinity stress in all treated plants may have multiple explanations, pointing to the parallel and overlapping mechanisms that control and modulate physiological responses to stress. Possibly the extensive utilization of AsA for the detoxification of H 2 O 2 , accompanied by the inefficient regeneration of ascorbate, as proposed by Barbieri et al. (2012) [31], may explain the decreased concentration at the end of the growth period. Overall, after 15 days of saline treatment, the protection that AsA confers to basil plants may be considered insufficient. It should be noted here that although the AsA concentration is correlated with salt tolerance, it is obviously not the only responsible substance, since other physiological mechanisms and metabolites, not determined in the present study, may also contribute.  High salinity interferes with uptake and assimilation of certain nutrients [37], mainly through alterations in related enzyme activity. The source of salinity; i.e., the composition and concentration of salts in the irrigation water or nutrient solution, significantly shapes the type and magnitude of nutrient-related problems; deficiencies, ion toxicities, and altered ion balance and competition may differentially arise due to various salinity sources [38]. In salinity-related research, the use of NaCl predominates, yet there are many other sources of excessive salts that may impact crops, and their result in plant nutritional response may be different [12,39]. In the context of cascade hydroponics, using nutrient solutions of increasing elemental concentration, thus increasing EC, is a more realistic approach compared to NaCl addition. The enhanced EC substantially modified the nutrient absorption and content. Nutrient imbalances were found in basil plants exposed to salinity in both harvests of the current study (Table 3), with the effect being more pronounced at the intermediate harvest (15 days). At the final harvest, the leaf elemental concentration may reflect the trade-off between enhanced nutrient availability in the irrigation solution and the salinity effects on plant function and metabolism; accordingly, we followed the interference of imposed salinity to nutrient status. Under T5, T10, and T15, N content in leaf tissues was increased at both harvest dates compared to the control. The opposite trend has also been reported by Elhindi et al. (2017) [23], but the differences may be ascribed to their use of NaCl for imposing salt stress, the longer duration of their experiment (57 days), and possibly to the different developmental stage of basil plants at their final harvest, since flowering alters nutrient allocation patterns. Indeed, NaCl-imposed salinity stress has profound effects on N concentration due to inhibition of NO3-transport systems [40]. Corroborating our results, Scagel et al. (2017) [39] found increased N content in basil High salinity interferes with uptake and assimilation of certain nutrients [37], mainly through alterations in related enzyme activity. The source of salinity; i.e., the composition and concentration of salts in the irrigation water or nutrient solution, significantly shapes the type and magnitude of nutrient-related problems; deficiencies, ion toxicities, and altered ion balance and competition may differentially arise due to various salinity sources [38]. In salinity-related research, the use of NaCl predominates, yet there are many other sources of excessive salts that may impact crops, and their result in plant nutritional response may be different [12,39]. In the context of cascade hydroponics, using nutrient solutions of increasing elemental concentration, thus increasing EC, is a more realistic approach compared to NaCl addition. The enhanced EC substantially modified the nutrient absorption and content. Nutrient imbalances were found in basil plants exposed to salinity in both harvests of the current study (Table 3), with the effect being more pronounced at the intermediate harvest (15 days). At the final harvest, the leaf elemental concentration may reflect the trade-off between enhanced nutrient availability in the irrigation solution and the salinity effects on plant function and metabolism; accordingly, we followed the interference of imposed salinity to nutrient status. Under T5, T10, and T15, N content in leaf tissues was increased at both harvest dates compared to the control. The opposite trend has also been reported by Elhindi et al. (2017) [23], but the differences may be ascribed to their use of NaCl for imposing salt stress, the longer duration of their experiment (57 days), and possibly to the different developmental stage of basil plants at their final harvest, since flowering alters nutrient allocation patterns. Indeed, NaCl-imposed salinity stress has profound effects on N concentration due to inhibition of NO 3 -transport systems [40].
Corroborating our results, Scagel et al. (2017) [39] found increased N content in basil leaves exposed to either NaCl-or CaCl 2 -induced salinity. N concentrations in basil in the present experiment, except for the increased availability in the irrigation solution, may also be related to the enhanced concentration of certain amino acids mentioned above. Indeed, the induction of glutamine and asparagine synthesis during stress has been linked to storage of organic nitrogen and transport within plants [33]. Phosphorus uptake was gradually decreased along increasing salinity (Table 3), a result that has been also found in NaCl-challenged basil [20,23]. According to Scagel et al. (2017) [39], the source of salinity determines the mechanism of P reduction, being either limited availability of phosphate ions or competition with other ions for binding sites within roots. Apparently, in the case of P in the current experiment, the salinity effect outweighed the increasing P supply by irrigation solution. Potassium content in leaves exhibited an interesting profile. T10 and T15 plants showed significantly lower K content compared to the control and T5 at the intermediate harvest, although their irrigation solution permitted increased K availability. An increase was evident during the last days, resulting in similar K levels in all treatments at the final harvest. This increase may be due to the role of K in osmoregulation, since it is considered, along with Cl, among the inorganic solutes with a greater contribution to the osmotic adjustment in basil [21]. Similar regulatory involvement in osmotic stress may be ascribed to Ca, the accumulation of which was induced by the two higher salinity levels at the early phase of stress. Ca content enhancement in leaves has been ascribed to its altered allocation under salt stress [40], which has been evidenced also in basil [39] as an increased translocation from root to shoot. The micronutrients determined in the present study showed distinct profiles along treatments and harvests (Table 3) [20] found that 5, 10, and 20 dS m −1 did not significantly alter Fe, Mn, and Zn concentrations. However, in an earlier work by the same authors [39], a substantially increased uptake of Cu and Zn was found, along with a reduced uptake of Fe with either NaCl-or CaCl 2 -imposed stress. Of course, the direct comparison with other works may be misleading, since it is documented that differences in salt source used and salinity tolerance among basil cultivars may account for specific effects on leaf nutrient composition [12,39].
In conclusion, the various aspects of basil growth and biochemical performance collectively indicated the 5 dS m −1 salinity level as the upper limit/threshold of tolerance to stress. Additionally, the results of the first experiment indicated the first 15 days of treatment as a critical point for the process of salinity-symptom appearance in growth performance, as well as mineral composition.

Is Basil Suitable as a Secondary Crop in a Cascade Hydroponics System?
The second experiment was established to evaluate the suitability of basil as a secondary crop in a cascade hydroponics system. The inevitably moderate to high electrical conductivity of the solution that drains from the primary crop to the secondary one challenges the growth and functional performance and depends on the salinity-tolerance thresholds of the latter. In the current experiment, basil grown in pots directly received the drainage solution of cucumber grown in hydroponics without any further treatment.
The growth response of basil clearly correlated with the low salinity level of the first experiment analyzed above. Even though the EC of the solution that was channeled to basil never exceeded 3.5 dS m −1 , a reduction of growth was evident, notably at the final harvest ( Figure 5). All aspects of growth were affected by salinity to a different extent, ranging from 20% reduction of the projected leaf area to 47% and 42% reduction of the fresh and dry weights, respectively ( Figure 5B-D). Similar growth restrictions were also recorded in the T5 plants in the first experiment (Figures 1 and 2). Elvanidi et al. (2020) [5] reported similar reductions of basil grown in cascade hydroponics in which basil received only 40% of cucumber drainage complemented with typical irrigation water.
stress. Additionally, the results of the first experiment indicated the first 15 days of treatment as a critical point for the process of salinity-symptom appearance in growth performance, as well as mineral composition.

Is Basil Suitable as a Secondary Crop in a Cascade Hydroponics System?
Τhe second experiment was established to evaluate the suitability of basil as a secondary crop in a cascade hydroponics system. The inevitably moderate to high electrical conductivity of the solution that drains from the primary crop to the secondary one challenges the growth and functional performance and depends on the salinity-tolerance thresholds of the latter. In the current experiment, basil grown in pots directly received the drainage solution of cucumber grown in hydroponics without any further treatment.
The growth response of basil clearly correlated with the low salinity level of the first experiment analyzed above. Even though the EC of the solution that was channeled to basil never exceeded 3.5 dS m −1 , a reduction of growth was evident, notably at the final harvest ( Figure 5). All aspects of growth were affected by salinity to a different extent, ranging from 20% reduction of the projected leaf area to 47% and 42% reduction of the fresh and dry weights, respectively ( Figure 5B-D). Similar growth restrictions were also recorded in the T5 plants in the first experiment (Figures 1 and 2). Elvanidi et al. (2020) [5] reported similar reductions of basil grown in cascade hydroponics in which basil received only 40% of cucumber drainage complemented with typical irrigation water.  The total amino acid content of basil that received the drainage solution from cucumber (hereinafter referred to as "treated plants") displayed a trend for higher values compared to the control only at the final harvest ( Figure 6). Accumulation of amino acids is usually connected to a stress-induced protein breakdown as mentioned above; nevertheless, plants may actively synthesize specific amino acids that play a distinct and beneficial role in stress response [33]. In this line, the 67% increase of glutamic acid concentration at the final harvest (Table 4) may be correlated with its use as a precursor to essential amino acids or its newly reported signaling role toward increased activities of antioxidative enzymes [41]. A trend toward increase in citrulline concentration in treated plants may be ascribed to its function as a compatible solute involved in the maintenance of cellular osmolarity [42]. Overall, the profile of amino acids of the treated plants was in accordance with the T5 basil plants in the first experiment. However, there was a significant difference in magnitude of certain amino acid responses between the T5 and the second experiment. For example, while glycine, ornithine, and proline had a 40-60% increase in treated plants compared to the control in the second experiment, their increase in T5 was two-to fivefold of the control values. Additionally, two-to sevenfold increases in asparagine, glutamine, and arginine of T5 plants were not found in the second experiment. The above-mentioned distinct responses indicated that other factors apart from EC might also act as drivers of the regulation of free amino acid homeostasis and control the dynamic amino acid pool. We may speculate that these factors were related to cucumber root exudates that enriched the drainage solution, and affected the basil plants' response, but they were not determined in the present study. Further and targeted experiments on exudate composition and their detailed metabolomic profile are needed to validate this hypothesis.
The ascorbic acid content of treated plants showed an increase compared to the control plants, but was statistically significant only in the intermediate harvest. The same trend was observed at the final harvest, but it was marginally non-significant (Figure 7). This finding was slightly different compared to the AsA concentration of T5 plants presented above ( Figure 4). It seemed that the drainage solution from cucumber triggered the antioxidant machinery, and the response was more pronounced during the acclimation process compared to the first experiment. Apparently, other antioxidants, not determined in the present study, may also play a role in this process. The total amino acid content of basil that received the drainage solution from cucumber (hereinafter referred to as "treated plants") displayed a trend for higher values compared to the control only at the final harvest ( Figure 6). Accumulation of amino acids is usually connected to a stress-induced protein breakdown as mentioned above; nevertheless, plants may actively synthesize specific amino acids that play a distinct and beneficial role in stress response [33]. In this line, the 67% increase of glutamic acid concentration at the final harvest (Table 4) may be correlated with its use as a precursor to essential amino acids or its newly reported signaling role toward increased activities of antioxidative enzymes [41]. A trend toward increase in citrulline concentration in treated plants may be ascribed to its function as a compatible solute involved in the maintenance of cellular osmolarity [42]. Overall, the profile of amino acids of the treated plants was in accordance with the T5 basil plants in the first experiment. However, there was a significant difference in magnitude of certain amino acid responses between the T5 and the second experiment. For example, while glycine, ornithine, and proline had a 40-60% increase in treated plants compared to the control in the second experiment, their increase in T5 was two-to fivefold of the control values. Additionally, two-to sevenfold increases in asparagine, glutamine, and arginine of T5 plants were not found in the second experiment. The above-mentioned distinct responses indicated that other factors apart from EC might also act as drivers of the regulation of free amino acid homeostasis and control the dynamic amino acid pool. We may speculate that these factors were related to cucumber root exudates that enriched the drainage solution, and affected the basil plants' response, but they were not determined in the present study. Further and targeted experiments on exudate composition and their detailed metabolomic profile are needed to validate this hypothesis.    Table 5 summarizes the macro-and micro-nutrient content of basil plants in the second experiment. Noticeable impacts of treatment were recorded in P concentration, with 40% and 52%, and Mn with 74% and 60% reductions in the intermediate and final harvests, respectively; and in K, which showed a significant 45% reduction at the end of the experiment. On the contrary, Mg concentration was increased by 108% at the final harvest.   Table 5 summarizes the macro-and micro-nutrient content of basil plants in the second experiment. Noticeable impacts of treatment were recorded in P concentration, with 40% and 52%, and Mn with 74% and 60% reductions in the intermediate and final harvests, respectively; and in K, which showed a significant 45% reduction at the end of the experiment. On the contrary, Mg concentration was increased by 108% at the final harvest. The performance of basil under the conditions and the system in which the present experiment was carried out proved to be promising for its use as a secondary crop in cascade hydroponic systems. Obviously, there are numerous aspects of basil biochemistry, complementary to those measured in the present study, that might be determined in future studies and complete the picture of basil performance. Among them, the impact of the drainage solution for various primary crops on concentrations and profiles of secondary metabolites, especially those responsible for aroma, would be worth studying.
The concept of cascade cropping systems is new; thus, few studies have explored their potential in ornamental and horticultural production and delineated their advantages and drawbacks [5,8,43,44]. The main constraint seems to be the increased salinity in the root zone of the secondary and tertiary crops, a problem that may be overcome by various levels of dilution of the primary crop leachates with water of low electrical conductivity [5,8,45]. Additionally, the use of salt-tolerant or even halophytic species, which can successfully grow under conditions of increased salinity, may be a feasible idea. Future experiments are expected to focus on this latter group, i.e., halophytes, some of which have recently been domesticated and included in human diet. Therefore, halophytes may be excellent candidates for their use as tertiary crops in cascade hydroponics.

Conclusions
The present study explored the salinity-tolerance thresholds of basil to evaluate its potential use as a secondary crop in a cascade hydroponics system. We used two distinct but complemented approaches to address our target; the first experiment tested several aspects of basil's response to increasing levels of salinity in order to identify the tolerance limits, while the second experiment employed a cascade system to monitor the responses, with cucumber grown in hydroponics as the primary crop, the drainage solution of which irrigated basil grown in pots, a setup comparable to the first experiment. The various aspects of basil growth and biochemical performance collectively indicated the 5 dS m −1 salinity level as the upper limit/threshold of tolerance to stress. Additionally, the results of the first experiment indicated the first 15 days of treatment as a critical point for the process of salinity-symptom appearance on growth performance, as well as mineral composition. The use of basil as a secondary crop, which inevitably faces increased EC of the drainage solution of the primary crop, is subject to a compromise between fresh produce reduction and an increase in specific biochemical attributes related to basil quality. The increase of total amino acids under enhanced EC in both experiments and the trend for higher levels of the antioxidant AsA, as a surrogate of the antioxidant pool of basil, may compromise the 40% reduction in fresh produce yield in the cascade system. Another important aspect that should be considered is the benefit of re-using the drainage solution from the primary crop, which results in combined production of more than one crop and the optimization of the environmental footprint. Comparing the two experiments reported in the current study, we should highlight certain different responses of basil's biochemical parameters when exposed to drainage solution in the cascade system. This finding may indicate that other factors, except for the increased EC, may also act as drivers of plant response, and this must be confirmed in future experiments to reach deeper insights. We concluded after both experiments that basil performed well under the specific conditions and in the system employed in the present study and might be a good candidate for use as a secondary crop in cascade hydroponics systems. Funding: This research has been co-financed by the European Union and Greek national funds through the national action "Bilateral and Multilateral E&T Cooperation Greece-Germany" (project code: T2DGE-0893); and for the German action, by the German Federal Ministry of Education and Research.