Interactive Effects of Salinity Stress and Irrigation Intervals on Plant Growth, Nutritional Value, and Phytochemical Content in Mesembryanthemum crystallinum L.

: Halophytes such as ice plants are concurrently subjected to salt and drought stresses in their natural habitats, but our knowledge about the effects of combined stress on plants is limited. In this study, the individual and combined effects of salinity and irrigation intervals on the plant growth, mineral content, and proximate and phytochemical composition of M. crystallinum


Introduction
As the world's population grows, so will global food production, putting additional pressure on already-scarce resources such as clean irrigation water and arable land [1,2].In addition, increasing soil salinity and dry conditions caused by climate change are regarded as the most critical and adverse environmental factors for plants, leading to enormous coir, and vermiculite (1:1:1), as described by Loconsole et al. [17].A layer of course bark was laid in the seed tray before the medium was added to prevent leaching.Approximately 2 kg of the medium was added to each tray.Thereafter, the seeds were evenly broadcasted on the trays, followed by the application of a thin layer of vermiculite on top of the seeds.The trays were then watered with Captab (4 g/L) manufactured by Universal Crop Protection (Pty) Ltd., Kempton Park, South Africa, to prevent the development of fungal diseases and were placed on a heating bed under mist irrigation sprayers for germination.After the emergence of the first set of true leaves, one hundred and ninety-two (192) germinated seedlings were washed using tap water to remove soil and other debris.They were then potted up in 12.5 cm black plastic pots containing river sand and, thereafter, were hardened off for a week in the greenhouse before they were moved to the experimental site.During this period, seedlings were irrigated daily with a nutritive solution formed by adding NUTRIFEED™ (manufactured by STARKE AYRES Pty. Ltd., Gauteng, South Africa) to municipal water at 10 g per 5 L. The nutrient solution contained the following ingredients: N (65 mg/kg), P (27 mg/kg), K (130 mg/kg), Ca (70 mg/kg), Cu (20 mg/kg), Fe (1500 mg/kg), Mo (10 mg/kg), Mg (22 mg/kg), Mn (240 mg/kg), S (75 mg/kg), B (240 mg/kg), and Zn (240 mg/kg).After acclimatisation, plants were watered with distilled water for 5 days to wash off any salt residue and, thereafter, were organized into four irrigation treatments ((1) 100 mL once a day; (2) 100 mL once every 2 days; (3) 100 mL once every 4 days; (4) 100 mL once every 8 days) with four salt concentrations (0, 200, 400, and 800 ppm) applied in each treatment.Salt concentrations were set up by adding increasing concentrations of NaCl to the nutrient solution and a handheld EC meter (Martini EC 59, manufactured by Milwaukee ® , Milwaukee, MI, USA) was used to measure the salt concentration in the nutrient solution.A completely randomized design was used to accommodate the two-way factorial experiment.Forty-eight (48) replicates per factorial combination were used, totalling to 192 plants for the entire experiment.

Determination of Plant Growth 2.3.1. Leaf Length and Number of Leaves
To determine new growth, the length and quantity of leaves were employed as variables.Every two weeks, the leaf length was measured using a 30 cm ruler from the substrate level to the tip of the tallest shoot, and leaf counting was performed manually.

Plant Weight
Shoots and roots were divided at the post-harvest stage, and the fresh weights of various samples were determined using a typical laboratory scale (RADWAG ® Model PS 750.R2).The plant material was subsequently dried to a consistent weight in an oven at 55 • C using a LABTECHTM model LDO 150F (Daihan Labtech India Pty. Ltd., 3269 Ranjit Nagar, New Delhi, India).The difference between the fresh and dry weights was compared with the amount of water held within plants' tissues.

Chlorophyll Content
The leaf SPAD readings (Chl SPAD ) were obtained from two fully formed leaves of each plant using a chlorophyll meter (SPAD-502, Konica Minolta, Japan).The readings/figures were averaged out by the SPAD-502 m to produce a final number.

Nutritional Analysis 2.5.1. Sample Preparation
Dried leaves of each set of replicates were pulverized with an electric motor blender and transferred into airtight containers, which were kept in a refrigerator at 4 • C for nutritional analyses.

Mineral Analysis
The elemental analysis was performed using the Inductively Coupled Plasma-Optical Emission Spectrometer in the analytical laboratory of the Department of Agriculture and Rural Development, KwaZulu Natal Province, South Africa, as described by Bulawa et al. [21], to determine the mineral composition of each set of replicates in the experiment.

Proximate Analysis Moisture Content
The procedure given by Jimoh et al. [22] with slight modifications was used to determine the moisture content.Empty porcelain vessels were dried in an oven at 105 • C for one hour, allowed to cool in a desiccator, and weighed W1.One gram of pulverised samples of M. crystallinum (W2) was placed in a vessel and oven-dried to a constant weight at 105 • C. The vessel and its contents were cooled in a desiccator before being reweighed (W3).The calculation below was used to determine the percentage of moisture content.
Following the recommendations and guidelines from the Association of Official Analytical Chemists (AOAC) [23], the crude fat was determined.A pulverized sample of about 1 g was extracted in 100 mL of diethyl ether and shaken on an orbital shaker for 24 h.The mixture was then filtered, and the filtrate was collected in previously weighed clean beakers.The ether extract was then equilibrated with 100 mL of diethyl ether and shaken for another 24 h on an orbital shaker, and the filtrate was collected in a beaker (W1).The ether filtrate was concentrated to dryness in a steam bath and oven-dried at 55 • C before being reweighed in the beaker (W2).The proportion of crude fat was calculated using the formula below.% Crude fat content = W2 − W1 original weight of the pulverised sample × 100

Ash Content
To calculate the percentage ash content of plant samples, the AOAC [23] technique was utilized.After being marked with sample codes using a heat-resistant marker, porcelain crucibles were oven-dried at 105 • C for one hour.The crucibles were weighed after cooling in a desiccator (W1).Thereafter, 1 g of ground samples was added to porcelain crucibles that had already been weighed (W2).The crucibles with the contents were placed in a muffle furnace set to 250 • C for 1 h and then 550 • C for 5 h to completely ash the samples.After desiccator cooling, the samples were weighed (W3).The samples' ash content was calculated as % Ash content = W2 − W3 W2 − W1 × 100

Crude Protein
Crude protein was determined by boiling 2 g of ground samples in a Kjeldahl flask with concentrated H 2 SO 4 (20 mL) until a clear mixture was obtained, with a digestion tablet acting as a catalyst.The digested extracts were distilled after being filtered and dissolved in 250 mL.An aliquot containing 50 mL of 45% NaOH was distilled further in a 500 mL round-bottomed flask, and 150 mL of the distillate was transferred into a flask containing 100 mL of 0.1 M HCl.This was then titrated with methyl orange against 2.0 mol/L NaOH.The endpoint of titration was indicated by a yellow colour change, and the percentage nitrogen content was calculated as shown in the equation below.

=
[(mL std acid × N of acid) − (mL bank × N of base)] − (mL std base × N of base) × 1.4007 original weight of the pulverised sample where N = normality, and the percentage crude protein was obtained by multiplying the nitrogen value by a constant factor of 6.25 (USDA, 2018).
Neutral Detergent Fibre (NDF) The NDF composition of the samples was determined using the equation below, as described by [22].C for 7 days.The dried material was ground into a fine powder using a Junkel and Kunkel model A 10 mill.The samples were extracted by mixing 100 mg of the dried powdered material with 25 mL of 80% (v/v) ethanol (Merck, South Africa) for 1 h.Thereafter, they were centrifuged at 4000 rpm for 5 min, and the supernatants were used for all analyses.

Total Polyphenols
The total polyphenol content of the extracts was performed using the Folin-Ciocalteu method as reported by [24] with slight modifications.About 25 µL of the sample was mixed with 125 µL of Folin-Ciocalteu reagent (Merck, Johannesburg, South Africa) that was diluted 10 times with distilled water in 96-well microplates.Thereafter, a 7.5% sodium carbonate solution was prepared and added to a 96-well microplate with extracts.The plate was incubated for 2 h at room temperature, and the absorbance was then measured at 765 nm in a Multiskan spectrum plate reader (Thermo Electron Corporation, Waltham, MA, USA).The standard curve was prepared using 0, 20, 50, 100, 250, and 500 mg/L gallic acid (Sigma, South Africa) in 10% EtOH, from which the polyphenolic content was extrapolated, and the results were expressed as mg gallic acid equivalent per g dry weight (mg GAE/g DW).

Estimation of Flavonol Content
The flavonol content of the extracts was determined using standard quercetin at 0, 5, 10, 20, 40, and 80 mg/L in 95% ethanol (Sigma-Aldrich, Johannesburg, South Africa) [25].A volume of 12.5 µL of the crude sample extracts was mixed with 12.5 µL 0.1% HCl (Merck, South Africa) in 95% ethanol and 225 µL of 2% HCl.The extracts were then incubated for 30 min at room temperature.The absorbance was read at 360 nm at a temperature of 25 • C. The results were expressed as milligram quercetin equivalent per gram dry weight (mg QE/g DW).

DPPH Free Radical Scavenging Activity
The DPPH radical was generated from a solution of 0.135 mM DPPH prepared in a dark bottle, as stated by Ohikhena et al. [26].A volume of 300 µL of DPPH solution was reacted with graded concentrations (0 and 500 µM) of Trolox standard (6-Hydrox-2,5,7,8tetramethylchroman-2-20 carboxylic acid) solution and 25 µL of crude extract.The mixtures were incubated for 30 min, after which absorbance was taken at 517 nM.The results were expressed as µM/Trolox equivalent per g dry weight (µM TE/g DW).

Ferric Reducing/Antioxidant Power (FRAP) Assay
The FRAP assay was performed using the method of [27,28].FRAP reagent was prepared by mixing 30 mL of Acetate buffer (0.3 M, pH 3.6) (Merck, South Africa) with 3 mL of 2,4,6-tripyridyl-s-triazine (10 mM in 0.1 M hydrochloric acid) (Sigma, South Africa), 3 mL of iron (III) chloride hexahydrate (FeCl 3 •6H 2 O) (Sigma, South Africa), and 6 mL of distilled water.In a 96-well plate, 10 µL of the crude sample extract was mixed with 300 µL of the FRAP reagent and incubated for 30 min at room temperature.The absorbance was then measured at 593 nm in a Multiskan spectrum plate reader (Thermo Electron Corporation, USA).The samples' FRAP values were calculated using an L-ascorbic acid (Sigma-Aldrich, South Africa) standard curve with concentrations varying between 0 and 1000 µM.The results were expressed as µM ascorbic acid equivalents (AAE) per g dry weight (µM AAE/g DW).

Statistical Analysis
Three samples from each treatment were examined for the mineral and proximate analyses, and all assays were performed in triplicate.The data are presented as mean values and standard errors (SE) and were analysed using two-way analysis of variance (ANOVA) followed by Tukey's least significant test at the p ≤ 0.05 significance level.The STATISTICA application, version 13.5.0.17, was used to conduct the analysis.

Leaf Length and Number of Leaves
The results obtained from this study showed that the M. crystallinum growth response to salinity and irrigation intervals was variable (Table 1).The interactive effect of salinity and irrigation intervals on leaf length was not statistically significant (p ≤ 0.05).However, this was not the case with the leaf number, where plants irrigated with 800 ppm salinity every four days had significantly higher leaf numbers than most treatments, including the control, but did not differ significantly from plants irrigated with 400 ppm salinity every four days.

Total Fresh Weight and Dry Weight
Salinity and irrigation intervals had a significant (p ≤ 0.05) effect on the total fresh weight and dry weight of M. crystallinum.The highest total fresh weight was obtained in plants irrigated with 800 ppm salinity every four days.This was significantly higher than the control and other treatments.The same trend was also observed in total dry weight: plants irrigated with 800 ppm salinity every four days had a higher dry weight than all treatments, including the control (Figures 1 and 2).Values (mean ± SE) followed by dissimilar letters in each column are significantly different at p ≤ 0.05 (*); ns = not significant.

Total Fresh Weight and Dry Weight
Salinity and irrigation intervals had a significant (p ≤ 0.05) effect on the total fresh weight and dry weight of M. crystallinum.The highest total fresh weight was obtained in plants irrigated with 800 ppm salinity every four days.This was significantly higher than the control and other treatments.The same trend was also observed in total dry weight: plants irrigated with 800 ppm salinity every four days had a higher dry weight than all treatments, including the control (Figures 1 and 2).

Effect of Salinity and Irrigation Intervals on Leaf Chlorophyll Content
The results revealed that salinity and irrigation intervals had a significant effect on the chlorophyll content of M. crystallinum leaves as the plant aged.At week 6, equivalent chlorophyll contents were recorded in all treatments, although at weeks 2, 4, and 8, the chlorophyll contents were variable, and the highest mean value of chlorophyll content was recorded in plants irrigated every four days without salinity treatment (Table 2).However, these values were comparable to those obtained in many treatments during the growing weeks, with the exception of week 8.In this last week, plants irrigated every four days without salinity had a higher chlorophyll content that was only comparable to that of plants irrigated every eight days without salinity.

Effect of Salinity and Irrigation Intervals on Leaf Chlorophyll Content
The results revealed that salinity and irrigation intervals had a significant effect on the chlorophyll content of M. crystallinum leaves as the plant aged.At week 6, equivalent chlorophyll contents were recorded in all treatments, although at weeks 2, 4, and 8, the chlorophyll contents were variable, and the highest mean value of chlorophyll content was recorded in plants irrigated every four days without salinity treatment (Table 2).However, these values were comparable to those obtained in many treatments during the growing weeks, with the exception of week 8.In this last week, plants irrigated every four days without salinity had a higher chlorophyll content that was only comparable to that of plants irrigated every eight days without salinity.Salinity and irrigation intervals had a significant effect (p < 0.05) on the accumulation of macronutrients in the leaves of M. crystallinum.The highest yield of nitrogen (2440 mg/100 g) was recorded in plants irrigated every eight days without salinity treatment (Table 3).This was significantly higher than in other treatments but was comparable to that of plants irrigated with 200 ppm salinity every eight days.On the contrary, the phosphorus composition of the samples was comparable among most treatments.The highest yield of 335 mg/100 g was recorded in plants irrigated every four days with 400 ppm salinity.Likewise, the highest yield of potassium (9855 mg/100 g) was also comparable to that in most treatments, including the control.Moreover, the highest yield of calcium was recorded in plants irrigated with 400 ppm salinity every four days, but this was comparable to that in most treatments, including the control.As for magnesium, the highest yield was recorded in plants irrigated every eight days without saline treatment.This was significantly higher than that in other treatments but was comparable to that in plants irrigated with 200 ppm every eight days.Values (mean ± SE) followed by dissimilar letters in each column are significantly different at p ≤ 0.05 (*); ns = not significant.

Micronutrients
Table 4 shows the amounts of accumulated heavy metals, such as zinc (Zn), manganese (Mn), copper (Cu), and iron (Fe), in the examined samples.This study revealed a significant increase in the accumulation of manganese in plants irrigated every eight days with or without saline treatment.The highest yield of manganese was recorded in plants irrigated every eight days without salinity.This was significantly higher than in the control and plants irrigated daily with 200, 400, and 800 ppm but was comparable to that in most treatments.This was not the case for iron, where the highest yield was recorded in plants irrigated every eight days with 200 ppm salinity.This was significantly higher than the control and other treatments.When assessing the accumulation of zinc, the highest yield was recorded in plants irrigated every eight days with 400 ppm salinity.This was comparable to that of plants irrigated every second, fourth, and eighth day without salinity and plants irrigated with 200 ppm every second and eighth day and 800 ppm every eight days.As for copper, the highest yield was recorded in control plants, but this was comparable to that of plants irrigated every two days without salinity and plants irrigated with 200 ppm salinity every two days.The highest sodium accumulation was recorded in plants irrigated with 800 ppm every four days but was comparable to that in most treatments.Values (mean ± SE) followed by dissimilar letters in each column are significantly different at p ≤ 0.05 (*); ns = not significant.

Effect of Salinity and Irrigation Intervals on Proximate Composition of Dried Leaves of M. crystallinum
This study discovered significant variations in the nutritional contents of M. crystallinum leaves under different salinity levels, irrigation intervals, and their interaction in terms of acid detergent fibre (ADF), ash, crude fat, moisture, neutral detergent fibre (NDF), and crude protein (Table 5).The highest acid detergent fibre was recorded in plants irrigated every two days without saline treatment, but this was comparable to that in plants irrigated daily, every four, and every eight days without salinity.Conversely, the treatments did not cause a significant variation in the ash, moisture, NDF, or protein contents of M. crystallinum leaves, as equivalent proximate nutrients were recorded for the treatments.When assessing crude fat within leaf samples, plants exposed to irrigation intervals only had higher crude fat values than those exposed to both salinity and irrigation intervals.The highest crude fat was recorded in control plants, but this was comparable to that in plants irrigated every two, four, and eight days without salinity and plants irrigated with 200 and 400 ppm salinity every eight days.Values (mean ± SE) followed by dissimilar letters in each column are significantly different at p ≤ 0.05 (*); ns = not significant.

Effect of Salinity and Irrigation Intervals on Phytochemicals and Antioxidant Activity of Dried
Leaves of M. crystallinum 3.5.1.Total Polyphenols Drought stress and its interactions with salinity had a significant impact on the accumulation of polyphenols in the leaves of M. crystallinum (Table 6).Plants exposed to drought stress only had a significantly higher yield of polyphenols when compared to plants exposed to both drought and salinity stress.The highest polyphenol content (20.06 mg GAE/L) was recorded in plants irrigated every eight days without saline treatment.This was significantly higher than in other treatments at p < 0.05 but was comparable to that of plants irrigated daily without salinity (Figure 3).Table 6.Two-way ANOVA F-Statistic for the effect of salinity and irrigation intervals on total fresh weight, total dry weight, total polyphenols, total flavonols, FRAP, and DPPH antioxidant capacity of M. crystallinum.

Total Flavonols
The same trend observed in total phenols was also observed in total flavonols within samples, where the highest yield was recorded in plants subjected to drought stress only.Plants irrigated every eight days without saline treatment had a significantly higher yield of flavonols when compared to plants subjected to both salinity and drought stress.Even though plants irrigated every eight days had a higher yield of flavonols, it was comparable to that in plants irrigated every four days without saline treatment (Figure 4, Table 6).The same trend observed in total phenols was also observed in total flavonols within samples, where the highest yield was recorded in plants subjected to drought stress only.Plants irrigated every eight days without saline treatment had a significantly higher yield of flavonols when compared to plants subjected to both salinity and drought stress.Even though plants irrigated every eight days had a higher yield of flavonols, it was comparable to that in plants irrigated every four days without saline treatment (Figure 4, Table 6).

FRAP Capacity
The total FRAP content in the leaves of M. crystallinum was significantly influenced by drought, salinity and their interaction at p ≤ 0.05 (Table 6).The highest FRAP capacity was recorded in plants irrigated every eight days without saline treatment.This was significantly higher than in most treatments but was comparable to that of the control,

FRAP Capacity
The total FRAP content in the leaves of M. crystallinum was significantly influenced by drought, salinity and their interaction at p ≤ 0.05 (Table 6).The highest FRAP capacity was recorded in plants irrigated every eight days without saline treatment.This was significantly higher than in most treatments but was comparable to that of the control, plants irrigated every eight days with 200 ppm, plants irrigated every eight days with 400 ppm and plants irrigated every eight days with 800 ppm salinity (Figure 5).

DPPH Capacity
The DPPH capacity in the leaves of M. crystallinum varied significantly at p ≤ 0.05 under varying salinity and drought stress compared with the control (Table 6).The highest DPPH capacity was recorded in plants irrigated every eight days without salinity compared to all treatments, including the control.This was significantly different from the control and most treatments but was comparable to the DPPH capacity of plants irrigated every eight days with 200 ppm salinity (Figure 6).

DPPH Capacity
The DPPH capacity in the leaves of M. crystallinum varied significantly at p ≤ 0.05 under varying salinity and drought stress compared with the control (Table 6).The highest DPPH capacity was recorded in plants irrigated every eight days without salinity compared to all treatments, including the control.This was significantly different from the control and most treatments but was comparable to the DPPH capacity of plants irrigated every eight days with 200 ppm salinity (Figure 6).

Discussion
Drought and salinity are known to be the most common coexisting factors affecting crop yield and productivity [29].Earlier reports have stated that the combined effects o drought and salinity are more detrimental than the individual effects of each stress on plant growth and yield [30,31].Although the patterns of different plants' responses to sa linity and drought are similar, their thresholds to stress vary from one species to another In M. crystallinum, the individual effects of drought and salinity have been extensively studied, and the results have shown that the plant is drought-and salt-tolerant [17,32] However, their combined effects have not yet been subject to much research.In this study, the individual and combined effects of salt and drought stress were evaluated on common ice plants.Results have shown that plants irrigated every two days without sa linity had a longer leaf length.However, this was comparable to that of plants subjected to salinity and drought, suggesting that the combined effects on leaf length were no significant.These findings concur with the results reported by Alam et al. [11] on Salsola imbricata (Fetid Saltwort), where the combined effect of salt and water stress on shoo length was not significant.However, these results contradict the findings of Calone et al [33] on Limonium angustebracteatum, where the individual effect of salt stress did no have a negative effect on leaf length but was remarkably affected by both factors.This suggests that the combined tolerance to drought and salinity could be species-specific among halophytes.When assessing the number of leaves among treatments, plants irri gated every four days with 800 ppm salinity had the highest number of leaves.This in crease also resulted in higher total fresh and dry weights.These results support the find ings of Sogoni et al. [24], who observed a significant increase in the leaf/branch number

Discussion
Drought and salinity are known to be the most common coexisting factors affecting crop yield and productivity [29].Earlier reports have stated that the combined effects of drought and salinity are more detrimental than the individual effects of each stress on plant growth and yield [30,31].Although the patterns of different plants' responses to salinity and drought are similar, their thresholds to stress vary from one species to another.In M. crystallinum, the individual effects of drought and salinity have been extensively studied, and the results have shown that the plant is drought-and salt-tolerant [17,32].However, their combined effects have not yet been subject to much research.In this study, the individual and combined effects of salt and drought stress were evaluated on common ice plants.Results have shown that plants irrigated every two days without salinity had a longer leaf length.However, this was comparable to that of plants subjected to salinity and drought, suggesting that the combined effects on leaf length were not significant.These findings concur with the results reported by Alam et al. [11] on Salsola imbricata (Fetid Saltwort), where the combined effect of salt and water stress on shoot length was not significant.However, these results contradict the findings of Calone et al. [33] on Limonium angustebracteatum, where the individual effect of salt stress did not have a negative effect on leaf length but was remarkably affected by both factors.This suggests that the combined tolerance to drought and salinity could be species-specific among halophytes.When assessing the number of leaves among treatments, plants irrigated every four days with 800 ppm salinity had the highest number of leaves.This increase also resulted in higher total fresh and dry weights.These results support the findings of Sogoni et al. [24], who observed a significant increase in the leaf/branch number and total fresh and dry weights in Tetragonia decumbens subjected to salinity and drought.This behaviour can be explained by the availability of epidermal bladder cells in the leaves of many halophytes, including the common ice plant [17].These bladder cells are known to have water and NaCl storage functions for osmotic adjustments in the vacuole, which enables these species to dilute excessive salt and enhance plant growth [17].
Drought and salt stress have been reported as major factors responsible for senescence mechanisms in plants, which cause a reduction in the chlorophyll content of many plant species [34].Nevertheless, the effect of these abiotic factors on chlorophyll reduction varies from species to species [35].In the present study, leaf chlorophyll contents among tested treatments were comparable to that of the control during the growing weeks and were only reduced in the last week before harvest.This reduction in chlorophyll content did not have any negative effects on plant growth.These findings were also observed by Atzori et al. [2] in a field experiment, where the common ice plant was not negatively affected by the reduction in the photosynthetic apparatus as the plant aged under increased seawater irrigation.These findings confirm that the species can tolerate both salt and drought stress under cultivation.
The nutritional quality of commercial crops around the world has been heavily affected by salinity and drought conditions, with a more pronounced effect in arid regions [36].This has triggered an increasing global interest in the investigation of the nutritional and nutraceutical value of halophytes to tackle malnutrition and increase food security in countries affected by drought and salinity [37].Halophytes have been proven to possess important healthy minerals with several phytochemical compounds essential for human consumption [38,39].In the present study, high yields of minerals present in the leaves of the common ice plant under salinity and drought were found to be comparable to those in other edible halophytes, such as Aster tripolium, Sarcocornia perennis, Salicornia ramosissima, and Arthrocnemum macrostachyum, which are already consumed and sold in restaurants and supermarkets in other parts of the world [34,38,40].The variation observed in the results shows that drought and salinity modulate mineral composition, since most minerals tested were higher than the recommended dietary intake allowance (RDA).For instance, 2000 mg of potassium is required for an adult, a limit that was obtained in all tested treatments, including the control.When compared to the previous literature on other leafy vegetables subjected to salinity and drought, such as New Zealand spinach, water spinach, Salicornia, and Sarcocornia, the common ice plant was shown to have a high content of potassium [1,41,42].This mineral is the most prevalent intracellular cation and plays a crucial role in excitable tissues such as the heart, neurons, and skeletal muscles, as it is essential for action potentials and electrical excitability [43].Therefore, the daily consumption of the leaves of the common ice plant would be a good source of dietary potassium.
The composition of magnesium in the analysed leaves of all treatments ranged from 340 to 1135 mg/100 g, and these values are higher than the RDA of 55 mg/100 g, proving that this species is a rich source of magnesium.The values attained in this study are comparable to those recorded by Patricia et al. [44] and Mih et al. [45] on wild vegetables consumed by the people of Lebialem Highlands, Southwestern Cameroon, and Northern Cote D'Ivoire.Magnesium is well known for preventing a number of illnesses, such as cardiovascular disorders, and its deficiency is also linked to the aetiology of diabetes mellitus [46,47].Additionally, it is needed in the human body as an intracellular electrolyte and as a co-factor for the creation of numerous enzymes, proteins, and nucleic acids [48].Thus, the consumption of this species will help in mitigating several illnesses.
When assessing the composition of calcium within the tested samples, most treatments meet the RDA of 1000 mg in respect of drought and salinity exposure.These results substantiate those obtained by Davis et al. [49] on Sambucus nigra exposed to varying environmental stresses, where the leaves and flowers had calcium that ranged from 500 to 1228 mg/100 g.The common ice plant has proven to be a good source of calcium, which has the ability to retain extracellular fluids, build bones and teeth, and transmit nerve impulses, blood clotting, and muscle contraction [50,51].However, when examining the phosphorus composition within samples, all treatments fell short of the RDA of 700 mg.These results are in agreement with the findings of Jimoh et al. [22] on Amaranthus caudatus, where the examined samples had lower phosphorus than the RDA of 700 mg.
The accumulation of sodium (Na) in the leaves and roots of many halophytes in bio-saline agriculture has been reported to cause a decline in cations such as K + , Ca 2+ , and Mg 2+ [42].In this study, none of these cations declined, supporting the existence of a clearcut nutrient absorption system that allows large NaCl compartmentation.This then enables the species to accumulate high Na content that is beyond the RDA of 200-500 mg proposed for healthy living organisms and might cause health problems.Caparrota et al. [52] recommended the boiling technique, as it reduced the Na composition of spinach leaves cultivated under seawater irrigation.Thus, it will be vital to cook or boil ice plant leaves before consumption when cultivated under saline conditions.
A variety of micronutrients, including iron, zinc, aluminium, copper, and manganese, are crucial for human nutrition [53].However, their daily consumption is required in small quantities of not less than 20 mg, which accounts for less than 0.01% of body weight [54].The micronutrients accumulated in the leaves of the ice plant were below the recommended daily allowance of 20 mg, except iron, which was above the RDA of 20 mg in all treatments, including the control.Iron has been reported to be the most commonly deficient micronutrient in school children, and its deficiency has been implicated in anaemia, fatigue, and blood-related diseases [55,56].Most people receive iron by eating vegetables, particularly spinach [39].Using spinach as a comparison, it is clear that ice plant leaves are a richer source of iron, displaying levels 5-10 times higher than those found in spinach.
When assessing the proximate composition of the leaves of the ice plant, variation among treatments were observed.Salinity and drought are known to impair plant nutrition; however, in this study, the ash content of the samples increased with increasing salinity and drought conditions.Ash has been used to measure the nutritional value of food and is believed to be an indicator of the mineral contents that have been conserved in food items.The ash content of the tested samples ranged from 35 to 52%, which is higher than the 5% reported for other wild vegetables and corresponds to the composition found in processed foods [57].These results concur with Ntuli's [53] research on two species of water spinach (Ipomea plebeian R.Br. and Ipomea wightii (Wall.)Choisy), where the ash level was reported to range from 20 to 38%.The plant's high ash value suggests that it is an abundant source of dietary fibre.
Most wild vegetables, including halophytes, have been reported to have low levels of unsaturated fats that range from 2 to 4% [21].The amount of crude fat in the analysed samples concurs with this finding of [21] since it was lower than 3% in all tested treatments, including the control.Excessive fat in food can lead to increased cholesterol, which is a major cause of cardiovascular disorders.Thus, the consumption of this leafy vegetable will be suitable for the management of weight loss and diseases caused by excess fat content.When assessing the protein content within samples, it ranged from 9 to 15%.This is similar to what was reported by Ajayi et al. [58] on Amaranthus cruentus (11.32%) and Solanum nigrum (15.06%).This implies that the ice plant can be a good natural source of protein and could reduce protein malnutrition in children, especially in developing nations.
Dietary fibre is essential for controlling bowel movements, preventing cardiovascular disease, and slowing the absorption of cholesterol.The neutral detergent fibre (NDF) present in the tested samples ranged from 20 to 29% and was higher than the values reported in other wild vegetables, such as Amaranthus cruentus (8.45%), Celosia argentea (23%), and Solanum nigrum (9.56%) [55,58].The NDF concentration found in ice plant leaves is a sign that the species may assist in regulating intestinal transit, increasing dietary bulk, and lowering the risk of various metabolic illnesses such as colon cancer, obesity, and diabetes that are brought on by the insufficient intake of crude fibre.Moreover, the tested samples possessed a lower moisture content that ranged from 7 to 10%, suggesting that the leaves of this species might have lower microbial contamination and chemical degradation, which are normally associated with high moisture content [59].These lower values imply that the leaves of the ice plant may have a lengthy storage life, benefiting producers and sellers.
Abiotic factors such as salinity and drought stress are known to increase the production of reactive oxygen species (ROS), triggering oxidative stress and the activation of antioxidant mechanisms in plants [60].During this process, many plant species alter their growth and produce metabolites, such as phenolic compounds, which act as reducing agents, hydrogen donors, and singlet oxygen quenchers [61].These phenolic compounds are of great intrinsic importance in human nutrition since they scavenge free radicals and suppress lipid peroxidation in human tissues, which prevents potential issues brought on by the excessive consumption of synthetic additives.In the present study, the individual effect of drought stress optimized the yield of polyphenols and flavonols, while its interaction with salinity reduced these compounds.These findings contradict those of Alam et al. [11] on Salsola imbricata (Fetid Saltwort) subjected to both salinity and drought, where an increase in proline accumulation acted as an antioxidative defence system to maintain a balance between ROS over-accumulation and their elimination to keep ROS at the signalling level required for plant growth [62].The positive effect of salinity in drought-stressed halophytes has also been reported in Atriplex halimus L., where the accumulation of antioxidants was reduced in samples subjected to the combined effect of salinity and drought [63].This improved drought tolerance under salinity may be due to osmotic adjustment through higher Na + and proline accumulation and antioxidative enzymes [64].Nevertheless, the leaves of ice plants subjected to both salinity and water stress possessed more polyphenols and flavonols than other promising edible halophytes in South Africa, such as Chenopodium album, Trachyandra divaricata, Trachyandra ciliata [21,[65][66][67], and Tetragonia decumbens [24,68].This suggests that the leaves of this plant may be a good source of nutritional antioxidants.
In summary, increased salt concentrations did not influence the high yields of acid detergent fibre (ADF), crude fat, protein, neutral detergent fibre (NDF), and phytochemicals and antioxidants in M. crystallinum, as these phytonutrients were recorded in high amounts in plants subjected to irrigation intervals only, although a combination of salinity and irrigation intervals resulted in the highest ash and moisture contents.These findings validate earlier reports that M. crystallinum can excrete salt through its foliar epidermal bladder, thereby normalizing salt-induced stress, which could trigger the increased accumulation of phytochemicals, antioxidants, and proximate nutrients in the species.

Conclusions
The results of this study show that salinity and irrigation intervals influence the production of phytochemicals, as well as vegetative development and nutritional value, in M. crystallinum leaves.Plants supplemented with 800 ppm salinity every four days revealed a significant increase in growth parameters, while the nutritional composition was comparable among treatments.The accumulated yields of phytochemicals, antioxidants, and nutritional components in the leaves of M. crystallinum are within the recommended daily allowances for consumption and reflect a nutrient-supplying vegetable crop.The plant's high fibre, ash, and protein contents attest to its value as an immune booster, a significant nutraceutical, and a possible functional food for humans.Additionally, the lower moisture level suggests that the leaves of this species may have a lengthy shelf life, benefiting producers and sellers.These findings suggest that M. crystallinum could provide an additional source of nutrients in regions affected by both salinity and irrigation intervals.Thus, its domestication in South Africa, a water-scarce nation, is highly recommended.

Figure 1 .Figure 1 .
Figure 1.Effect of salinity and irrigation intervals on total fresh weight of M. crystallinum.Means (bars) that share the same letter do not vary significantly according to Tukey's test (p ≤ 0.05).

Figure 2 .
Figure 2. Effect of salinity and irrigation intervals on total dry weight of M. crystallinum.Means (bars) that share the same letter do not vary significantly according to Tukey's test (p ≤ 0.05).

Figure 2 .
Figure 2. Effect of salinity and irrigation intervals on total dry weight of M. crystallinum.Means (bars) that share the same letter do not vary significantly according to Tukey's test (p ≤ 0.05).3.3.Effect of Salinity and Irrigation Intervals on the Mineral Content of Dried Leaves of M. crystallinum 3.3.1.Macronutrients

Figure 3 .Figure 3 .
Figure 3.Effect of salinity and irrigation intervals on total polyphenols of M. crystallinum.Means (bars) that share the same letter do not vary significantly according to Tukey's test (p ≤ 0.05).3.5.2.Total Flavonols

Figure 4 .
Figure 4. Effect of salinity and irrigation intervals on total flavonols of M. crystallinum.Means (bars) that share the same letter do not vary significantly according to Tukey's test (p ≤ 0.05).

Figure 4 .
Figure 4. Effect of salinity and irrigation intervals on total flavonols of M. crystallinum.Means (bars) that share the same letter do not vary significantly according to Tukey's test (p ≤ 0.05).

24 Figure 5 .
Figure 5.Effect of salinity and irrigation intervals on FRAP capacity of M. crystallinum.Means (bars) that share the same letter do not vary significantly according to Tukey's test (p ≤ 0.05).

Figure 5 .
Figure 5.Effect of salinity and irrigation intervals on FRAP capacity of M. crystallinum.Means (bars) that share the same letter do not vary significantly according to Tukey's test (p ≤ 0.05).

Figure 6 .
Figure 6.Effect of salinity and irrigation intervals on DPPH capacity of M. crystallinum.Mean (bars) that share the same letter do not vary significantly according to Tukey's test (p ≤ 0.05).

Figure 6 .
Figure 6.Effect of salinity and irrigation intervals on DPPH capacity of M. crystallinum.Means (bars) that share the same letter do not vary significantly according to Tukey's test (p ≤ 0.05).

Table 1 .
Effect of salinity and irrigation intervals on leaf length and number of leaves of M. crystallinum.

Table 2 .
Effect of salinity and irrigation intervals on chlorophyll content of M. crystallinum leaves.

Table 2 .
Effect of salinity and irrigation intervals on chlorophyll content of M. crystallinum leaves.
Values (mean ± SE) followed by dissimilar letters in each column are significantly different at p ≤ 0.05 (*); ns = not significant.

Table 3 .
Effect of salinity and irrigation intervals on macronutrients of M. crystallinum leaves.

Table 4 .
Effect of salinity and irrigation intervals on micronutrients of M. crystallinum leaves.

Table 5 .
Effect of salinity and irrigation intervals on proximate composition of M. crystallinum leaves.

Two-Way ANOVA F-Statistic Total Fresh Weight Total Dry Weight Total Polyphenols Total Flavonols FRAP Capacity DPPH Capacity
(*) Indicates significant difference at p ≤ 0.05.