Assessment of different salt concentrations on the growth and phytochemical change of the ice plants

2.1 Plant growth conditions

During the summer of 2021, the current investigation was conducted in the controlled plant factory at Gyeongsang National University, Smart Farm Systems Laboratory, South Korea. The total duration of the experiment was 120 days (from early March to late June). The main environmental parameters, namely temperature, humidity, and CO₂ concentration, were monitored daily using a specific high precision sensor unit (Hanam Engineering Co. ltd, South Korea) (Madhavi et al., 2022).

Seeds of the ice plant (Mesembryanthemum crystallinum L.) were sown in 50 cell plug trays [(54 cm × 28 cm × 4 cm (L × W × H) ] using bio plus compost soil (cocopeat 68.86%, peat moss 11.00%, perlite 11.00%, and zeolite 9.00%) as a growing medium (Madhavi et al., 2022). Germinated seedlings were grown in a plant factory system for 28 days at 25 ± 1 °C, 60 ± 10% humidity, 1000 µmolmol ⁻¹ CO₂, and under 120 µmolm⁻²s⁻¹ photosynthetic photon flux density (PPFD) using red, blue, and green LED treatment (Hanam Engineering Co. ltd, South Korea) with 16 h/8h (light/dark) photoperiod as shown in Fig. 1.

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Fig. 1

Seedling stage (30 days after planting) of M. crystallinum grown under the plant factory system.

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Plants were watered three times a week with 50 ml of 0.5 × Hoagland’s solution (Hoagland and Arnon, 1950). After appearing 4 leaves (28 days mature plant), 9 seedlings per treatment were transplanted in bio plus compost containing pots [(60 cm × 20 cm × 12 cm (L × W × H)] (Guan et al., 2020). For each treatment, 3 pots were used for 9 plants (20 cm apart; 3 plants per pot), with the single plant representing an independent biological replicate.

The different number of replicates were utilized for evaluating the effect of salt treatments on morphological growth, such as canopy area in the juvenile stage (n = 3), and the number of leaves, lateral stems, SPAD value, fresh weight, and dry weight of shoot and root in the adult stage (n = 9). Eventually, the cation and anion contents and DPPH radical scavenging activity(%) in the adult stage, three replicates were used.

Following the protocol by Cushman et al., 1990, plants in the control group were watered daily with 50 ml of 0.5 × Hoagland’s solution (application into one pot; 3 plants), while those in the treatment group were also watered daily with 0.5 × Hoagland’s solution containing the different salt treatments per pot as demonstrated in Table 1 (Guan et al., 2020). Previous studies reported that 400 mM NaCl exhibited the highest growth performance and bioactive component accumulation (Agarie et al., 2009). In this study, individual CaCl₂ concentrations were selected as 100 mM, 200 mM, and 400 mM as previous studies' NaCl individual concentrations (Agarie et al., 2007; Agarie et al., 2009). In addition to that, NaCl and a mixture of CaCl₂ and NaCl solution concentrations were maintained up to 400 mM in each salt treatment, and electrical conductivity (EC) was evaluated. EC was maintained at the same level for each solution, as denoted in Table 1. Initially, salt solutions were prepared and monitored EC (“Measuring soil salinity: plant stress, n.d.”). Simultaneously, the salt solution (25 ml) was mixed with 0.5 × Hoagland’s solution (25 ml) and applied to the pot, as mentioned previously. EC values of final salt solutions with 0.5 × Hoagland’s solution were indicated in Table 1. Eventually, the influence of salt stress on ice plant growth, and phytochemical changes were measured. The facts mentioned above affect the selection of eight salt treatments for our study.

2.1.1

2.1.1 Measurements of plant growth and morphology

Plant morphological parameters, namely number of leaves, number of lateral stems, and, SPAD value, were measured during 120 days of mature (adult phase) plants. Typically, the canopy area was high in the early stage, decreasing the later growth stage of all treatments. Therefore, canopy area measurement in the juvenile stage is crucial. Otherwise, all leaves drop and become small in the reproductive stage, as revealed by Bohnert and Cushman, 2000. The canopy area was measured during 60 days (juvenile phase) of mature plants by creating a python program with the green pixels against the reference reported by Guan et al., 2020.

The number of leaves was counted for all plant leaves except the cotyledons in each of the nine plants per treatment. The number of lateral stems was counted in every-nine plants. A portable chlorophyll meter (SPAD-502, Konica Minolta Inc., Osaka, Japan) was used to measure the value SPAD, indicating the chlorophyll content of the third leaf pair from the bottom leaf pair, in each of the nine plants per treatment (Kim et al., 2018). Subsequently, the canopy area was determined using the image processing algorithm developed by python program from Google Colaboratory using the ratio of green pixels against 4 cm² purple square reference paper with images taken by RGB camera (SONY DSC-RX100 vii, Korea) (Chaudhary et al., 2012). Three plant images were photographed in each control and salt treatment group, as demonstrated in Fig. 2. Each image of three plants was taken at 2 pm on Friday to track canopy growth and use the python program to measure canopy area (Fig. 2). Each image was captured using the RGB camera, cropped, and downscaled to a ratio of 0.25; the green, yellow and cyan colour threshold was extracted using the HSV colour model and converted to a black and white image shown by Madhavi et al., 2021. Precisely, plant and reference black and white image pixel values were calculated separately and evaluated the canopy area relevant to the reference area.

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Fig. 2

Images acquisition for the canopy area measurement (60 days mature plant) of M. crystallinum grown under the plant factory system. The purple-coloured square paper demonstrated the reference for calculating the canopy area.

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Eventually, the plants (120 days mature plant) were harvested three months after salt application. Fresh weights of shoots and roots were measured immediately after harvest using a digital balance (Model-FX-300iWP, A&D Company Limited, Tokyo, Japan). The dry weights were determined after drying the tissues in a drying oven (Shelves for 5E-DHG6310: 2 layers, Changsha Kaiyuan Instruments Co., ltd, Changsha 410100, PR China) at 80 °C for 48 h (Agarie et al., 2009). Generally, all weight measurements were taken in every-nine plants per treatment.

2.2 Ion content determination

2.3 DPPH radical scavenging activity(%)

Dried ice plant leaves (1 g) were ground with a mortar and pestle and placed in a centrifuge tube (15 ml) with 5 ml of 80% methanol. The mixture was shaken at room temperature for 12 h and centrifuged at 10,509×g for 10 min. After centrifugation, the supernatant was filtered through Whatman 120 MM no 1 filter paper (Lee et al., 2015). The 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich Co., MO, USA) determined the free radical scavenging activity of the ice plants modified method described by Kim et al 2021. The filtrates were used for analyzing the free radical scavenging activity. The reaction mixture containing 1 ml of extract and 3 ml of 0.3 mM DPPH was shaken vigorously for 5 s and incubated in the dark at room temperature for 20 min. After incubation, the absorbance of the extract was measured at 517 nm using an Ultraviolet-VIS-NIR Spectrophotometer (U-4100, Hitachi, America), with methanol as the blank and methanol and DPPH solution as the control (Weeplian et al., 2018). The scavenging effect for DPPH radical was calculated using the following equation:

Scavenging effect (%) = (1- A_s/A_c) × 100 (Weeplian et al., 2018).

A_s and A_c absorbance of sample and absorbance of the control respectively, at 30 min.

2.4 Statistical analysis

Data on morphology, ion content, and DPPH radical scavenging of ice plants were collected in MS Excel (Microsoft Office 2019, Seattle, WA, USA). Moreover, for data analysis, statistical methods in the Statistical Package for the Social Sciences (SPSS) were used, including analysis of variance (one-way ANOVA) to practice with a significance level of p < 0.05. Significant differences between the means of the experimental data were tested using a Post- Hoc Tukey's HSD test in Statistics 10 (SPSS version: 25.0.0, IBM, New York, USA). All graphical plots were illustrated using OriginPro 9.0 (Origin Lab Corporation Northampton, MA, USA).

3.1 Measurements of plant growth and morphology

Ice plants grown under different salt treatments showed appreciable differences in morphological parameters such as number of leaves, number of lateral stems, SPAD value, fresh and dry weight of shoots and roots, and canopy area. Subsequently, the number of leaves was significantly higher in treatment T₄, whereas there was less leaf production with T₅ and T₆ treatments, as shown in Table 2. Consequently, higher leaf production was observed in CaCl₂ treatments. This is because calcium plays a direct role in the regulatory mechanisms that activate plants under salt stress and enhance the stimulation of growth regulators and other substances in plants (Xu et al., 2013). However, the CS, T_1, T₂, T₃, and T₇ showed no significant differences in total leaf numbers due to the different concentrations of NaCl and CaCl₂ salt treatments.

Correspondingly, the number of lateral stems was promoted by T₄ and T₇ treatments, whereas lateral stem production was suppressed by the T₆ treatment compared to other salt treatments (Table 2). Bohnert et al., 2000 found that this species is a salt accumulator, storing a high amount of NaCl and CaCl₂ in a gradient from roots to developing shoots. Furthermore, at adequate salinity levels ranging from 100 to 400 mM, this salt-loving plant grew at its best (Agarie et al., 2007). Therefore, lateral stem growth was stimulated by the high concentration of salt treatment.

Interestingly, fresh weight and dry weight of shoots and roots tended to be higher in treatments T₄ and T₇, as shown in Table 2. Furthermore, a positive correlation was observed with the number of leaves, lateral stems, and canopy area related to the ice plant's shoot and root biomass. Kim et al., 2021 also exhibited that the number of leaves, lateral stems, and leaf area of ice plants positively correlated with the root and shoot biomass. Thus, it concluded that the highest morphological growth is appreciably related to the biomass of ice plants. Agarie et al., 2007 reported that 400 mM NaCl increased the dry weight of wild-type ice plants by almost 2-fold compared to the mutant ice plants. Therefore, the higher concentration of salt impacts the biomass enhancement of ice plants.

Different salt concentrations affected the SPAD value and photosynthetic rate inside the ice plant leaves (Table 2). Moreover, the SPAD value was significantly higher in T₆ than within the other different treatments. The photosynthetic rate was not appreciably different within the salt treatments CS, T₁, T₄, T_5, and T₇. A low photosynthetic rate was observed in salt treatments T₃ and T₂. However, the SPAD value and photosynthetic rate of ice plant leaves showed a considerably high value in T₆ treatment because the similar concentration of NaCl and CaCl₂ increased chlorophylls and net photosynthetic rate (Trajkova et al., 2006). The higher concentration of NaCl and CaCl₂ decreased the contents of chlorophylls and the net photosynthetic rate. It accelerates the respiration rate and CO₂ compensation concentration in leaves, as revealed by Khavari-Nejad and Chaparzadeh (1998).

The T₄ (258.08 ± 2.09 cm²) and T₇ (207.74 ± 1.21 cm²) salt treatments produced a larger canopy area during the early growth stage, while the T₃ treatment produced a lower canopy area (60.26 ± 2.57 cm²). Besides, salt application induces cell expansion and protein synthesis in ice plants, facilitating leaf expansion and a high growth rate (Yang and Yen, 2002). The response of canopy area for the other salt treatments in the juvenile stage was observed in T₂ (183.01 ± 2.47 cm²) followed by T₁ (174.78 ± 3.41 cm²), T₅ (133.11 ± 2.52 cm²), T₆ (103.68 ± 2.06 cm²), and CS (66.36 ± 1.42 cm²) as in Fig. 4. There were no appreciable differences between CS and T₃ during the early stage of the slow uptake of salt to plant (Figs. 3, 4).

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Fig. 3

HSV developed images for calculating the canopy area of ice plants in their juvenile growth stage.

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Fig. 4

The impact of various salt treatments on the canopy area of juvenile stage ice plants (60-days mature plants). The data indicate the means ± SD (n = 3). Different letters above bars indicate significant differences at p < 0.05.

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3.2 Ion content determination

3.2.1

3.2.1 Cation content determination

The results of cation content clearly showed that the ice plant accumulated comparatively much Na⁺ in T₄ (210.72 ± 9.24 mg g⁻¹) and less in T₁ (89.58 ± 1.33 mg g⁻¹) was expressed on a dry weight basis (Fig. 5A). There were no significant differences for Na⁺ storage in ice plant leaves among T₅ and T₇ treatments and between T₁ and T₂ salt treatments. The Na⁺ storage in ice plant leaves response to other salt treatments were T₄ (210.72 ± 9.24 mg g⁻¹), T₆ (204.08 ± 6.80 mg g⁻¹), T₇ (187.34 ± 8.97 mg g⁻¹), T₅ (181.06 ± 9.43 mg g⁻¹), T₃ (156.91 ± 4.12 mg g⁻¹), CS (104.40 ± 2.89 mg g⁻¹) and T₂ (93.69 ± 3.02 mg g⁻¹) in respectively. Furthermore, T₄ and T₆ exhibited the highest Na⁺ content due to the salt transport from root cells into leaf mesophyll relying on myo-inositol, and sodium interaction between myo-inositol facilitates the sodium uptake and long-distance transport. Moreover, the synergy between Ca²⁺ ion accumulation leads to the synthesis of myo-inositol and the uptake of Na⁺ (Nelson et al., 1999). As a result, sodium is stored in EBCs and plant younger tissues, and pinitol production in the ice plant shoot top is enhanced by salt. A similar tendency was observed in the Ca²⁺ ion results, as demonstrated in Fig. 5C.

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Fig. 5

The effect of different salt treatments on cation content of ice plant leaves in the adult stage (120 days mature plant). Data indicate the means ± SD (n = 3). Different letters above bars indicate significant differences at p < 0.05. (A) Na⁺, (B) K⁺, (C) Ca²⁺, (D) Mg²⁺ indicated the ion content measurement based on dry weight, respectively.

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According to the K⁺ ion results, the highest value was measured in T₃ (118.23 ± 5.26 mg g⁻¹), and the lowest value CS (33.04 ± 2.46 mg g⁻¹) represented as a percentage of dry weight basis, respectively. There was no appreciable difference noted among K⁺ ion accretion among T₄ and T₅ treatments, T₆ and T₇ treatments, and T₁ and T₂.

Based on the K⁺ ion results the remaining treatment showed the storage such as T₅ (103.37 ± 2.75 mg g⁻¹), T₄ (98.36 ± 5.13 mg g⁻¹), T₇ (95.62 ± 4.35 mg g⁻¹), T₆ (93.20 ± 5.36 mg g⁻¹), T₂ (46.50 ± 2.52 mg g⁻¹) and T₁ (43.40 ± 1.82 mg g⁻¹) respectively. These results have revealed that a lower concentration of CaCl₂ influences the accumulation of K⁺ ions in tissues of leaves and EBCs.

Moreover, increasing NaCl up to 400 mM reduced the K⁺ ion accumulation of photosynthetically active leaf tissues of ice plants, as illustrated in Fig. 5B. However, considering the Ca²⁺ ion, the significantly high concentration existed in T₄ in dry weight basis (20.90 ± 0.90 mg g⁻¹) and less in T₂ (5.19 ± 0.61 mg g⁻¹) respectively (Fig. 5C). No significant difference was observed in the Ca²⁺ ion accumulation among T₃ and T₅ treatments, and CS, T_1, and T₂ treatments. According to the results, Ca²⁺ ions in other salt treatments were observed T₆ (17.08 ± 1.57 mg g⁻¹), T₅ (14.21 ± 1.06 mg g⁻¹), T₃ (13.44 ± 1.04 mg g⁻¹), T₇ (11.01 ± 0.30 mg g⁻¹), T₁ (5.72 ± 0.79 mg g⁻¹), CS (5.62 ± 0.98 mg g⁻¹) dry weight basis in consecutively. Thus, results showed an increasing trend in salt treatments and accumulation of Ca²⁺ ions in leaf tissues in ice plants.

Regarding the Mg²⁺ ion, the highest accumulation was observed in T₃ as a dry weight basis (11.62 ± 1.06 mg g⁻¹) and the lowest in CS (3.90 ± 0.53 mg g⁻¹) orderly (Fig. 5D). Based on the results no significant difference was observed among T₃, T₅ (11.54 ± 0.33 mg g⁻¹) and T₆ (10.89 ± 0.17 mg g⁻¹) treatments and between T₄ (9.37 ± 0.93 mg g⁻¹) and T₇ (9.09 ± 0.78 mg g⁻¹) treatments and among T₁ (4.41 ± 0.60 mg g⁻¹), and T₂ (4.13 ± 0.48 mg g⁻¹) treatments in respectively. According to the results mentioned above, Na⁺ and Ca²⁺ ions showed a positive tendency for accumulation. In contrast, similar trends were observed K⁺ and Mg²⁺ ions deposition in ice plant leaves. However, Na⁺, K^+, and Mg²⁺ absorption were high in the presence of CaCl₂ salt treatment due to the NaCl suppressing the K⁺ ion concentration and CaCl₂ enhancing the leaf Cl⁻ and Ca²⁺ level, which leads to the uptake of other cations (Trajkova et al., 2006). A high concentration of NaCl worked on the lack of absorption of K⁺, Mg^2+, Ca²⁺, also Na⁺ competes with K⁺ and replaces it in the cell, as reported by Mohamed and Basalah, 2015.

3.2.2

3.2.2 Anion content determination

To assess the possible content of anion sequestration on a dry weight basis, the Cl⁻ stored high in T₁ (57.35 ± 0.63 mg g⁻¹) and less in CS (26.06 ± 1.96 mg g⁻¹) consecutively. According to the anion storage in ice plant leaves, no significant differences were observed among T₁ and T₅ (57.28 ± 3.58 mg g⁻¹) treatments and T₄ (35.74 ± 2.73 mg g⁻¹), T₆ (38.46 ± 1.63 mg g⁻¹) and T₇ (37.23 ± 2.00 mg g⁻¹) treatments. Following the Cl⁻ sequestration appreciable difference was observed T₃ (47.45 ± 2.18 mg g⁻¹) and T₂ (31.45 ± 1.24 mg g⁻¹) treatments in orderly. According to the individual (NaCl) and combination effect (NaCl and CaCl₂) results, the highest concentration, 400 mM, exhibited the highest leaf Cl⁻ accumulation compared to the lower concentration of salt treatment (T₂) and control samples, as shown in Fig. 6A.

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Fig. 6

The effect of different salt treatments on the anion content of adult stage ice plant leaves (120 days old plant). The data indicate the means ± SD (n = 3). Different letters above bars indicate significant differences at p < 0.05. (A) Cl⁻, (B) NO₃^–, (C) PO₄³⁻, (D) SO₄²⁻ denoted the ion content measurement based on dry weight, respectively.

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Correspondingly, NO₃^– and SO₄²⁻ are vital for amino acid synthesis and metabolic reactions of halophytic plants. According to the NO₃^– ion results from the highest value obtained in T₆ as dry weight basis (165.88 ± 1.63 mg g⁻¹) and lowest results followed by T₁ (90.58 ± 1.77 mg g⁻¹), T₇ (83.82 ± 2.65 mg g⁻¹) and T₅ (75.08 ± 4.99 mg g⁻¹) respectively (Fig. 6 B). However, no significant difference was shown among CS (120.12 ± 4.23 mg g-1), T₂ (124.58 ± 2.90 mg g⁻¹) and T₄ (124.76 ± 3.11 mg g⁻¹) treatments, and T₃ (75.69 ± 2.01 mg g⁻¹) and T₅ (75.08 ± 4.99 mg g⁻¹) treatments in respectively.

Concurrently, a similar pattern was discovered in SO₄²⁻ ion accumulation. The maximum value for SO₄²⁻ storage in leaves tissue was observed in T₃ (24.08 ± 2.73 mg g⁻¹) and the minimum in T₅ (9.23 ± 0.38 mg g⁻¹) on a dry weight basis orderly (Fig. 6 D). Based on the results, there were no significant differences among CS (17.89 ± 1.94 mg g⁻¹), T₁ (17.73 ± 1.84 mg g⁻¹), T₂ (18.99 ± 2.15 mg g⁻¹), T₇ (18.34 ± 0.97 mg g⁻¹) and T₄ (15.43 ± 2.20 mg g⁻¹) treatments in respectively.

Changes in NO₃^– and SO₄²⁻ concentrations in the leaves of the ice plant confirmed a comparable absorption trend (Fig. 6 B, D). Increased Cl⁻ concentrations competed with NO₃– uptake, and accumulation of SO₄²⁻. Generally, NO₃– and SO₄²⁻ showed the same ion exchange and sorption pattern described by Stefan et al., 2014. Furthermore and even more importantly, PO₄³⁻ is vital to plant growth and involved in several plant functions such as photosynthesis, the transformation of sugars, energy transfer, and nutrient movement, as reported by Mullins (2009). Therefore, elucidating the relationship between the energy state and stress tolerance PO₄³⁻ ion is vital for growth hormone presumably gibberellin production and regulates the abiotic stress in halophytes (Zhu et al., 2012).

Based on the PO₄³⁻ ion deposition results, the appreciable differences depicted T₃ as dry weight basis (80.41 ± 1.57 mg g⁻¹) followed by T₅ (52.84 ± 1.47 mg g⁻¹), T₇ (46.92 ± 3.06 mg g⁻¹), and T₂ (28.37 ± 3.73 mg g⁻¹) respectively (Fig. 6 C). Nonetheless, there was no significant difference obtained for the PO₄³⁻ ion deposition among in T₄ (41.58 ± 3.52 mg g⁻¹), CS (39.24 ± 2.83 mg g⁻¹), T₁ (36.17 ± 3.02 mg g⁻¹), T₆ (33.10 ± 2.40 mg g⁻¹) in consecutively.

As aforementioned, the results have revealed that a lower concentration of CaCl₂ significantly influences the PO₄³⁻ deposit in ice plant leaves.

3.3 DPPH radical scavenging activity(%)

The DPPH assay was used to assess the scavenging activity of ice plants under various salt stresses, as presented in Fig. 7. The lower value indicates the higher antioxidant activity, and the value of DPPH radical scavenging activity denoted the negative correlation with the antioxidant activity (Agarie et al., 2009). However, a scavenging activity among the salt treatments, no appreciable difference was denoted among T₅ (89.38 ± 1.21%), T₇ (87.96  ± 2.33%), CS (87.05  ± 5.23%) and T₄ (86.35  ± 4.88%) treatments and T₃ (45.98  ± 3.96%) and T₂ (44.93  ± 3.70%) treatments. There was significant radical scavenging activity was observed among T₆ (77.90  ± 2.10%), T₁ (52.59  ± 3.11%), T₂ treatments respectively.

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Fig. 7

The effect of different salt treatments on DPPH radical scavenging activity(%) of ice plants in the adult stage (120 days old plant). Data indicate the means ± SD (n = 3). Different letters above bars indicate significant differences at p < 0.05.

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According to the results, higher amounts of phenolic compounds are markedly promoted in the T₂ salt treatment, which has a minimum radical scavenging effect.

Salt stress was successful in promoting pinitol/ononitol accumulation in M. crystallinum halophytic characteristics. It was speculated that the combined salt created a turgor gradient along the axes of the mature ice plant, which resulted in the accelerated growth of new cells. Photosynthesis and photorespiration were associated with the concentration gradient from the root to the mesophyll cell. The methylation of myo-inositol to the intermediate ononitol produces pinitol, which appears to be epimerized to pinitol. These phenolic chemicals are primarily responsible for the colour, flavour, and aroma of plants, and they are also high in antioxidant activity (Hichri et al., 2011). A similar observation was obtained from the present study from T₁ treatment as low radical scavenging activity and high antioxidant activity by decomposition of phenolic compounds.

Antioxidant activity plays a fascinating role in removing reactive oxygen species that are harmful to the human body. According to Kim et al., 2021, it helps to avoid chronic diseases and ageing. As a result, several efforts to synthesize particular phytochemicals with antioxidant activity have been conducted. The bioactive concentrations in ice plants in T_1, T_2, and T₃ treatments were high due to the mild concentration of CaCl_2, and the high NaCl concentration affects the biosynthetic pathway through the accumulation of phenolic compounds. Based on the results, the combined impact of NaCl and CaCl₂ and a higher concentration of CaCl₂ were not affected by the increased antioxidant material as fewer secondary metabolites accumulated (Agarie et al., 2009).

Specifically, Agarie et al., 2009 revealed that the radical scavenging activity determined by the DPPH assay and the antioxidant activity of ice plants increased twofold by treatment with 400 mM NaCl, which was twice that of the lettuce plants. This study obtained a similar tendency of high antioxidant activity from the high concentration of NaCl and low CaCl₂ salt treatments such as T_1, T_2, and T₃. These results indicate the high potential of ice plants as polyol-rich, highly functional foods.