1 Introduction

Water spinach (Ipomoea aquatica Forsk; Convolvulaceae) is a common aquatic plant that grows as both an aquatic (floating over a freshwater surface) and terrestrial plant (Mandal et al. 2008). In China, water spinach was reported to be used as a green leafy vegetable in China Dynasty during 290–307 A.D. (Edie and Ho 1969; Austin 2007). The micronutrients present as 87 mg g−1 DW thiamine (vitamin B1), 1.37 mg g−1 AsA (ascorbic acid or vitamin C), 0.6 mg g−1 nicotinic acid, 120 g g−1 riboflavin and carotenoid (provitamin A), and the mineral content included 41.4 mg g−1 potassium (K), 31 mg g−1 magnesium (Mg), 5 mg g−1 sodium (Na), 2 mg g−1 calcium (Ca), and 1.7 mg g−1 zinc (Zn) per 100 g of water spinach (Mandal et al. 2008). In addition, major non-enzymatic antioxidants, α-tocopherol (vitamin E; 2.6 mg per 100 g) and AsA (50 mg per 100 g) have also been reported in the leaf tissues of water spinach (Ching and Mohamed 2001; Ismail and Fun 2003) that function as free radical-scavenging antioxidant compounds (Huang et al. 2005; Prasad et al. 2005). Recently, the microgreen vegetable in water spinach is a good candidate for mineral and phytochemical composition (Yadav et al. 2019). Microgreen production requires a large number of seeds with a high germination rate (> 80%; Ebert and Wu 2019). Therefore, the high density (biomass), uniformity plants, and best quality (enriched nutrients and phytochemical compounds) of water spinach in the mature stage still need further investigation.

In higher plants, NaCl salt elicitor is known to effectively promote secondary metabolites (Giri and Zaheer 2016) such as anthraquinone (alizarin and purpurin) in Rubia tinctorum (Aşcı et al. 2018); solasodine in Solanum nigrum (Bhat et al. 2008; Šutković et al. 2011); solamargine and solasonine in S. incanum (Al-Sinani and Eltayeb 2015); α-tocopherol in Carthamus tinctorius (Chavan et al. 2011); scopolamine in Datura metel (Ajungla et al. 2009); reserpine in Rauvolfia tetraphylla (Anitha and Kumari 2006); and salvianolic acid A, salvianolic acid B, and rosmarinic acid in Salvia miltiorrhiza (Yu et al. 2019). Low exposure to NaCl (50 mM) can promote the production of α-tocopherol, anthocyanins, and calcium mineral content in water spinach (Kitayama et al. 2019). Moreover, a combination of chemical elicitors such as NaCl and paclobutrazol (PBZ) can further affect the production of secondary metabolites as seen in Ocimum basilicum (Keramati et al. 2016) and Oryza sativa cv. KDML105 (Jasmine rice; Detpitthayanan et al. 2019). Paclobutrazol (PBZ) [(2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1H-1, 2, 4-trizol-1-yl)-pentan-3-ol] is an anti-gibberellic member of triazole family, which causes growth retardation in higher plants (Berova and Zlatev 2000; Desta and Amare 2021) and ameliorates abiotic stress effects (Soumya et al. 2017; Chandra and Roychoudhury 2020). The basic knowledge on the regulation of secondary metabolites in water spinach using chemical elicitors, NaCl and PBZ, is still lacking. The objective of this investigation was to promote vitamin E (tocopherol), vitamin C (AsA), calcium (Ca2+), total soluble sugar (TSS), physiological adaptation, and overall growth performances in two cultivars of water spinach using PBZ pretreatment and NaCl salt elicitor.

2 Materials and Methods

2.1 Plant Materials and PBZ/NaCl Treatments

Seeds of Chia Tai (CT; green stem variety) and wild type (WT; red stem variety) of water spinach were sown in the plastic bags (4 × 4 × 5 cm in W × L × H) containing 1 kg garden soil (EC = 2.69 dS m−1; pH = 5.7; total organic carbon = 12.26%; available N = 0.30 mg kg−1; available P = 578 mg kg−1; available K = 3,073 mg kg−1), a silt–clay soil class and cultivated under greenhouse conditions (80–90% relative humidity, 28–32 °C air temperature, 800–1000 mol m−2 s−1 photosynthetic photon flux density), according to Kitayama et al. (2019). Slow-releasing fertilizer (13–13-13 NPK) 10 g plant−1 was supplied by mixing into the soil substrate before the transplanting process. Three seeds were sown in each bag containing soil substrate, abnormal seedlings were thinned, and then the uniform plants in terms of size and number of leaves were selected as plant material. Six-week-old uniform seedlings with 4–5 true leaves (5.0 ± 0.5 cm in plant height) were foliar-sprayed only one time with 0 (control), 17, and 51 M paclobutrazol (PBZ) foliar application (50 mL plant−1) and then cultivated in the greenhouse for 1 week. Subsequently, water spinach seedlings pretreated by PBZ were subjected to 0 (control) and 100 mM NaCl (chemical elicitation) by flood-irrigation method for 10–15 min daily and were then cultivated in a greenhouse for 7 days. Overall growth performances, photosynthetic abilities, soluble sugar, tocopherol, ascorbic acid, Na+, and Ca2+ were measured in the treated plants.

2.2 Morphological Measurements

Plant height, number of leaves, leaf length, root fresh weight, shoot fresh weight, root dry weight, and shoot dry weight in each treatment were measured. Shoot and root were separated and dried in a hot air oven at 80 °C for 72 h, and subsequently transferred to a desiccant chamber until room temperature is achieved for dry weight determination.

2.3 Physiological Analysis

Photosynthetic pigments including chlorophyll a (Chla), chlorophyll b (Chlb), and total chlorophyll (TC) in the leaf tissues of water spinach were determined according to the method suggested by Shabala et al. (1998). One hundred milligrams of fresh leaf tissues was chopped, transferred to a glass vial containing 10 mL of 99.5% acetone, and then blended using a homogenizer. The glass vials were capped and sealed with Parafilm® to prevent evaporation, and then stored at 4 °C for 48 h. Chla and Chlb were measured at 662 nm and 644 nm, respectively, using a UV–VIS spectrophotometer (HACH DR/4000; Model 48,000, HACH Company, Loveland, Colorado, USA) against acetone (99.5%) as a blank.

Chlorophyll a fluorescence emission from the adaxial surface of a second fully expanded leaf was detected by a fluorescence monitoring system (FMS 2; Hansatech Instruments Ltd., Norfolk, UK) in the pulse amplitude modulation mode (Loggini et al. 1999). A target leaf, kept in the dark for at least 30 min, was initially exposed to the modulated measuring beam of far-red light (LED source) with a typical peak at a wavelength of 735 nm. Original (F0) and maximum (Fm) fluorescence yields were measured under weak modulated red light (1500 μmol m−2 s−1 PPFD) and calculated using FMS software for Windows®. The variable fluorescence yield (Fv) was calculated using the equation: Fv = Fm − F0. The ratio of variable to maximum fluorescence (Fv/Fm) was calculated as the maximum quantum yield of PSII photochemistry. The photon yield of PSII (ΦPSII) in the light was calculated as: ΦPSII = (Fm′ − F)/Fm′ after 45 s of illumination when a steady state was achieved (Maxwell and Johnson 2000).

Net photosynthetic rate (Pn; μmol m−2 s−1), transpiration rate (E; mmol H2O m−2 s−1), and stomatal conductance (gs; mmol m−2 s−1) of a second fully expanded leaf of water spinach were measured using a portable photosynthesis system (LI 6400XT, LI-COR, Lincoln, NE, USA) according to the method suggested by Cha-um et al. (2006). The air-flow rate of the IRGA chamber was fixed at 500 µmol s−1, and the chamber temperature was set at 27 °C. The light intensity was adjusted to 1000 µmol m−2 s−1 PPFD of 6400-02B red-blue LED light source.

2.4 Biochemical Assays

Soluble sugars (sucrose, glucose, and fructose) in the stem and leaf tissues were assayed following the method of Karkacier et al. (2003). γ- and α-Tocopherol in the stem and leaf tissues of water spinach were extracted and analyzed using the HPLC system (Fig. S1) following the method of Annunziata et al. (2012). Ascorbic acid in the stem and leaf tissues of water spinach was detected by the HPLC system (Fig. S2) according to the method suggested by Radulescu et al. (2013). Na+ and Ca2+ in root, stem, and leaf tissues were determined according to a protocol adopted from Tanaka et al. (1999) and Hossain et al. (2006).

2.5 Experiment Layout and Statistical Analysis

The experiment was designed as 3 × 2 × 2 factorials in a completely randomized design (CRD) with 5 replications (n = 5) of each treatment. The mean values were compared using Tukey’s HSD and analyzed by SPSS statistics software version 11.5. Pearson’s correlation coefficient was used for the validation of physiological, morphological, and yield traits.

3 Results

3.1 Growth Performances

In cultivar (cv.) CT, the dark green color of leaves was observed with an increase in PBZ concentration; however, plants under 100 mM NaCl for 7 days demonstrated a yellow color of leaves (Fig. 1). In contrast, the dark green color in the leaves of cv. WT was retained in each treatment (Fig. 1). Plant height in cv. CT was unchanged in all treatments (Fig. 2a and b) but, in cv. WT, declined by 16.5% under 17 M PBZ treatment and 46.6% under 51 M PBZ treatment over the control (0 M PBZ treatment). The plant height further dropped by 33.7% in 0-µM PBZ plants and 37.4% in 17-µM PBZ-pretreated plants when exposed to 100 mM NaCl (Fig. 2a) compared to control (0 mM NaCl). In addition, plant height in the 51-µM PBZ-pretreated WT plant was the shortest (58.07 cm) and did not vary significantly (43.50 cm) when subjected to 100 mM NaCl (Fig. 2a). In this case, the water spinach cvs. CT and WT were identified as PBZ unresponsive and responsive cultivars, respectively (Fig. 2c). The number of leaves in cv. CT was unaffected, while it was significantly dropped by 40.9% in 0-µM PBZ plants in cv. WT, when exposed to 100 mM NaCl (Table 1). Leaf length in cv. CT was significantly increased by 142% in 0-µM PBZ-pretreated plants, and in contrast, it was unchanged in cv. WT when exposed to 100 mM NaCl (Table 1). Root fresh weight in the cv. CT 0-µM PBZ treatment was sensitive to salt treatment, leading to a 34.5% decrease over the control, whereas it remained unaffected in cv. WT (Table 1). Shoot fresh weight in cv. CT 0-µM PBZ-pretreated plants and exposed to 100 mM NaCl sharply declined by 57.4% over the control (Table 1).

Fig. 1
figure 1

Plant morphological characteristics of water spinach (Ipomoea aquatica) cvs. Chia Tai (CT, commercial cultivar; green stem) and wild type (WT cultivar, red stem) pretreated with 0, 17, and 51 M paclobutrazol (PBZ) for 7 days, and subsequently subjected to 0 and 100 mM NaCl for 7 days

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Fig. 2
figure 2

Plant height of water spinach (Ipomoea aquatica) cvs. Chia Tai (CT, commercial cultivar; green stem) and wild type (WT cultivar, red stem) pretreated with 0, 17, and 51 M paclobutrazol (PBZ) for 7 days, and subsequently subjected to 0 and 100 mM NaCl for 7 days (a), the relationship between PBZ concentrations and plant height of CT (b), and WT (c). Data presented as mean ± SE (n = 5). Different letters in each column show significant differences at p ≤ 0.01, according to Tukey’s HSD

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Table 1 Number of leaves (NL), leaf length (LL), root fresh weight (RTFW), and shoot fresh weight (STFW) of two water spinach (Ipomoea aquatica) cultivars, Chia Tai or ‘CT’ and Wild Type or ‘WT’ seedlings, pretreated with 0, 17, and 51 M paclobutrazol (PBZ) and subsequently exposed to 0 and 200 mM NaCl for 7 days. Data in each column is represented as ± SE (n = 5)
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3.2 Na+ and Ca2+ Enrichment

Na+ content in the leaf, stem, and root tissues of water spinach significantly increased (root > stem > leaf) within 7 days of 100 mM NaCl salt treatment in both cvs. CT and WT, especially in the 0-µM PBZ treatment, when compared with untreated plants (0 mM NaCl) (Fig. 3a - c). However, Na+ levels in the PBZ-pretreated plants increased less rapidly compared to PBZ-untreated plants (Fig. 3c). Na+ contents in the stem and leaf tissues of both water spinach cultivars under 100 mM NaCl were significantly low in 51-µM PBZ-pretreated plants (Fig. 3a, b). On the other hand, Ca2+ contents in the leaf tissues of water spinach cv. CT were nearly the same in all the treatments, whereas it was found to increase in cv. WT pretreated with 17 and 51 M PBZ and subsequently exposed to 100 mM NaCl (Fig. 3d). In stem tissues, Ca2+ contents in both cvs. CT and WT pretreated with 51 M PBZ and subjected to 100 mM NaCl increased (Fig. 3e). Interestingly, Ca2+ in the root tissues of two water spinach cultivars increased with the PBZ and NaCl application rate (Fig. 3f).

Fig. 3
figure 3

Na+ in leaf (a), stem (b), and root tissues (c), Ca.2+ in leaf (d), stem (e), and root tissues (f) of water spinach (Ipomoea aquatica) cvs. Chia Tai (CT, commercial cultivar; green stem) and wild type (WT cultivar, red stem) pretreated with 0, 17, and 51 M paclobutrazol (PBZ) for 7 days, and subsequently subjected to 0 and 100 mM NaCl for 7 days (a), the relationship between PBZ concentrations and plant height of CT (b) and WT (c). Data presented as mean ± SE (n = 5). Different letters in each column show significant differences at p ≤ 0.01, according to Tukey’s HSD

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3.3 Photosynthetic Abilities and Relationships

Chla, Chlb, and TC content in water spinach cv. CT was sensitive to 100 mM NaCl, leading to their degradation by 22.1%, 50%, and 36% over the control, respectively. The pigments in NaCl-treated plants of cvs. CT and WT were sustained when pretreated with 51 M PBZ (Table 2). In cv. WT, Chlb and TC levels in NaCl-treated plants in the 0-µM PBZ application sharply reduced by 27.3% and 20.9% over the control, respectively. This led to a diminished quantum yield of PSII (Fv/Fm) and photon yield of PSII (ΦPSII) by 11.0% and 15% over the control, respectively (Table 2). In cv. CT, the net photosynthetic rate (Pn) in NaCl-treated plants in the 0-µM PBZ application was decreased by 41.1% over the control and it was improved by 17- and 51-µM PBZ foliar applications (Table 3). Pn, gs, and E in cv. WT in each treatment were greater than in cv. CT (Table 3).

Table 2 Chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll (TC), the maximum quantum yield of PSII (Fv/Fm), and photon yield of PSII (ΦPSII) two water spinach (Ipomoea aquatica) cultivars, Chia Tai or ‘CT’ and Wild Type or ‘WT’ seedlings, pretreated with 0, 17, and 51 M paclobutrazol (PBZ) and subsequently exposed to 0 and 200 mM NaCl for 7 days. Data in each column is represented as ± SE (n = 5)
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Table 3 Net photosynthetic rate (Pn), stomatal conductance (gs), and transpiration rate (E) of two water spinach (Ipomoea aquatica) cultivars, Chia Tai or ‘CT’ and Wild Type or ‘WT’ seedlings, pretreated with 0, 17, and 51 M paclobutrazol (PBZ) and subsequently exposed to 0 and 200 mM NaCl for 7 days. Data in each column is represented as ± SE (n = 5)
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A negative correlation between Na+ in roots and root fresh weight of cv. CT (Fig. 4a; R2 = 0.534), Na+ in roots and root fresh weight of cv. WT (Fig. 4b; R2 = 0.981), Na+ in shoots and shoot fresh weight of cv. CT (Fig. 4c; R2 = 0.387), and Na+ in shoots and shoot fresh weight of cv. WT (Fig. 4d; R2 = 0.832) was demonstrated. Moreover, a negative relationship between Na+ in leaves and TC content was found in cv. CT (Fig. 5a; R2 = 0.720), whereas it was independent in cv. WT (Fig. 5b). In contrast, a positive connection between TC content and ΦPSII was demonstrated in cv. CT (Fig. 5c; R2 = 0.666), while it was not observed in cv. WT (Fig. 5d). Moreover, positive relations between Chla content and Fv/Fm in cvs. CT (Fig. 6a; R2 = 0.512) and WT (Fig. 6b; R2 = 0.509) as well as ΦPSII and Pn in cvs. CT (Fig. 6c; R2 = 0.809) and WT (Fig. 6d; R2 = 0.272) were observed.

Fig. 4
figure 4

Relationships between Na+ in roots and root fresh weight of water spinach (Ipomoea aquatica) cvs. Chia Tai (CT, commercial cultivar; green stem) (a) and wild type (WT cultivar, red stem) (b), Na.+ in shoots and shoot fresh weight of CT (c) and WT (d), pretreated with 0, 17, and 51 M paclobutrazol (PBZ) for 7 days, and subsequently subjected to 0 and 100 mM NaCl for 7 days. Data presented as mean ± SE (n = 5)

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Fig. 5
figure 5

Relationships between Na.+ in leaves and total chlorophyll content of water spinach (Ipomoea aquatica) cvs. Chia Tai (CT, commercial cultivar; green stem) (a) and wild type (WT cultivar, red stem) (b), chlorophyll a content and maximum quantum yield of PSII (Fv/Fm) of CT (c) and WT (d), pretreated with 0, 17, and 51 M paclobutrazol (PBZ) for 7 days, and subsequently subjected to 0 and 100 mM NaCl for 7 days. Data presented as mean ± SE (n = 5)

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Fig. 6
figure 6

Relationships between total chlorophyll content and photon yield of PSII (ΦPSII) of water spinach (Ipomoea aquatica) cvs. Chia Tai (CT, commercial cultivar; green stem) (a) and wild type (WT cultivar, red stem) (b), ΦPSII and net photosynthetic rate (Pn) of CT (c) and WT (d), pretreated with 0, 17, and 51 M paclobutrazol (PBZ) for 7 days, and subsequently subjected to 0 and 100 mM NaCl for 7 days. Data presented as mean ± SE (n = 5)

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3.4 Soluble Sugar, AsA, and Tocopherol

Sucrose, glucose, and fructose contents in the leaf tissues of water spinach cv. CT grown under salt elicitor were increased by 908%, 1,623%, and 232%, respectively, over the control, but were subsequently reduced by PBZ foliar spray (Table 4). In the stem tissues, glucose was found to be enriched, while sucrose was declined by 35.3% and 51.9% over the control in 17- and 51-µM PBZ-pretreated plants, respectively, when plants were exposed to 100 mM NaCl (Table 4). In cv. WT, sucrose, and glucose in the leaf tissues of water spinach grown under salt elicitor decreased by 71.8% and 63.7% over the control, respectively, but these were significantly increased by PBZ pretreatment. In contrast, sucrose and fructose were elevated by 190% and 200% over the control, respectively, in 51-µM PBZ-pretreated plants exposed to NaCl treatment (Table 4). Total soluble sugar (TSS) in the leaf tissues of water spinach cv. CT was very low when not exposed to NaCl, but subsequently increased by 840%, 260%, and 470% over the control when pretreated with 0, 17, and 51 M PBZ, respectively, together with 100 mM NaCl (Fig. 7a). On the other hand, TSS declined in cv. WT plants in the 0-µ PBZ pretreatment and increased by 170% over the control in 51-µM PBZ-pretreated plants when exposed to 100 mM NaCl (Fig. 7a). TSS in stem tissues of water spinach cv. CT pretreated with 0 and 17 M PBZ and grown under salt elicitor were increased by 360% and 140%, respectively, over the control. In addition, TSS in stem tissues of cv. WT in was stable in all the treatments (Fig. 7d).

Table 4 Sucrose (Suc), glucose (Gluc), and fructose (Fruc) in leaves and stems of two water spinach (Ipomoea aquatica) cultivars, Chia Tai or ‘CT’ and wild type or ‘WT’ seedlings, pretreated with 0, 17, and 51 M paclobutrazol (PBZ) and subsequently exposed to 0 and 200 mM NaCl for 7 days. Data in each column is represented as ± SE (n = 5) 0963 8757 246
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Fig. 7
figure 7

Total soluble sugar (TSS) in leaf tissues (a), ascorbic acid in leaf tissues (b), tocopherol in leaf tissues (c), total soluble sugar (TSS) in stem tissues (d), ascorbic acid in stem tissues (e), tocopherol in stem tissues (f) of water spinach (Ipomoea aquatica) cvs. Chia Tai (CT, commercial cultivar; green stem) and wild type (WT cultivar, red stem) pretreated with 0, 17, and 51 M paclobutrazol (PBZ) for 7 days, and subsequently subjected to 0 and 100 mM NaCl for 7 days. Data presented as mean ± SE (n = 5). Different letters in each column show significant differences at p ≤ 0.01, according to Tukey’s HSD

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AsA in the leaf tissues of cv. CT grown under 100 mM NaCl was increased by 110%, 120%, and 110% over the control with 0-, 17-, and 51-µM PBZ pretreatments, respectively (Fig. 7b). In contrast, AsA in the stem tissues of cv. CT grown under 100 mM NaCl was significantly declined by 13.0%, 20.7%, and 8.3% over the control, in relation to 0-, 17-, and 51-µM PBZ pretreatments, respectively (Fig. 7e). In cv. WT, AsA in the leaf tissues of plants in 0-µM PBZ pretreatment was significantly declined when exposed to salt treatment and then it was steady in PBZ-treated plants (Fig. 7b). Moreover, AsA in the stem tissues of plants pretreated with 0- and 17-µM PBZ plants grown under 100 mM NaCl was increased by 130% over the control, while it was dropped by 17.5% over the control in 51-µM PBZ-treated plants (Fig. 7e). Tocopherol content was in the order leaf tissues > stem tissues, and it was higher in cv. WT as compared to cv. CT (Fig. 7c and d). Tocopherol in leaf tissues of cv. CT-pretreated with 0 and 17 M PBZ grown under 100 mM NaCl was increased by 120% and 130% over the control, respectively. In addition, tocopherol in both the cultivars treated with 51 M PBZ and 100 mM NaCl was promoted by 120% over the control (0 mM NaCl) (Fig. 7c). Degradation of tocopherol in stem tissues of two water spinach cultivars by salt elicitor was observed, except in the CT plants pretreated with 51 M PBZ (Fig. 7f). In the leaf tissues of cv. CT, γ-tocopherol was enriched by salt elicitor, especially in plants without PBZ pretreatment (174% over the control) and in plants with 17-µM PBZ pretreatment (183% over the control), and α-tocopherol was upregulated by salt elicitor (119% over the control) in plants pretreated with 51 M PBZ (Table 1). Likewise, γ-tocopherol in the stem tissues was enriched by salt elicitor (187% over the control; 51 M PBZ) (Table 5). In contrast, γ-tocopherol in NaCl-treated stem tissues of cv. WT pretreated with 0 and 17 M PBZ declined by 21.8% and 22.8% over the control, respectively, (Table 5). However, α-tocopherol was increased by 124% over the control in plants pretreated with 17 M PBZ and by 119% over the control in plants pretreated with 51 M PBZ. In stem tissues, only γ-tocopherol in NaCl-treated stem tissues of cv. WT pretreated plants with 51 M PBZ declined by 61.7% over the control, whereas it was maintained in other treatments.

Table 5 γ-Tocopherol and α-tocopherol of two water spinach (Ipomoea aquatica) cultivars, Chia Tai or ‘CT’ and wild type or ‘WT’ seedlings, pretreated with 0, 17, and 51 M paclobutrazol (PBZ) and subsequently exposed to 0 and 200 mM NaCl for 7 days. Data in each column is represented as ± SE (n = 5)
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4 Discussion

In general, the mode of action of PBZ is well-known as an anti-gibberellin plant growth regulator (GA-biosynthesis inhibitor), and therefore, it leads to a slow rate of shoot elongation (Kang et al. 1985; Yim et al. 1997; Forghani et al. 2018). However, the shoot height retardation rate depends on the cultivar, degree of PBZ dose, and their interaction (Lee et al. 2015; Lyons et al. 2018). In NaCl-treated plants, overall growth performances were retained by PBZ pretreatment (Hajihashemi et al. 2007; Khunpon et al. 2017). Therefore, plant height was significantly decreased with the treatment of PBZ and NaCl in sweet sorghum (Forghani et al. 2018). The reduction in Na+ content in each organ of salt-treated plants using PBZ application is still unclear. Likewise, low Na+ accumulation was observed in the root, stem, and leaf tissues of salt-treated peach (Prunus persica cv. Nemaguard) pretreated with 1000–2000 mg L−1 PBZ (El-Khashab et al. 1997). Similar results have also been observed in mango (Mangifera indica; Kishor et al. 2009), quinoa (Chenopodium quinoa; Waqas et al. 2019), oleander (Nerium oleander; Bañón et al. 2005), and wheat (Triticum aestivum; Hajihashemi et al. 2007). A low Na+ level in root and leaf tissues of salt-treated plants and Kinnow (Citrus nobilis × C. deliciosa) budded on Jatti Khatti (C. jambhiri) with 100 mg L−1 PBZ pretreatment was detected, leading to less toxicity in the plants as identified by high membrane stability index (MSI) (Kakade et al. 2014). In addition, Ca2+ enrichment has been observed with PBZ and salt treatment in water spinach. Previously, a Ca2+ level in water spinach was found to be alleviated by NaCl elicitor (Kitayama et al. 2019) and significantly regulated by PBZ (Kitayama et al. 2018). Moreover, Ca2+ in the leaf tissues of salt-treated citrus rootstock (Citrus karna) significantly declined, whereas it increased with the PBZ pretreatment (Sharma et al. 2011).

Photosynthetic abilities in NaCl-treated plants are good parameters to identify the plant’s ability to regulate stress and resultant toxic effects. Upon PBZ pretreatment, Chla, Chlb, and TC in salt-treated plants were maintained as compared to PBZ-untreated plants (Kishor et al. 2009; Khunpon et al. 2017; Forghani et al. 2018). In two Chinese bayberry genotypes (Myrica rubra cvs. Wangdao and Shenhong), Chla, Chlb, and TC in pretreated plants exposed to NaCl treatment were sustained (Hu et al. 2017). Similarly, degradation of chlorophyll pigment and stomatal function [gs and CO2 assimilation (A)] was prevented in PBZ-pretreated citrus rootstock plants (Citrus karna) grown under salt treatment (Sharma et al. 2011). In addition, Pn, gs, and E in salt-treated quinoa were significantly improved by PBZ pretreatment (Waqas et al. 2019). Previously, it has been reported that the degradation of Chlb and TC levels in two water spinach cultivars under salt treatment for 14 days was prevented by pretreatment (Kitayama et al. 2018).

Previously, Na+ enrichment in the leaf tissues of salt-treated plants damaged photosynthetic pigments, especially Chlb and TC, and consequently negative effects on overall photosynthetic abilities have been observed (Kitayama et al. 2019). A positive relationship between TC (Chla + Chlb) and Pn in two Chinese bayberry genotypes, Wangdao (R2 = 0.94) and Shenhong (R2 = 0.85), pretreated with PBZ and subsequently exposed to NaCl treatment was observed (Hu et al. 2017). Alternatively, malondialdehyde (MDA) content, a member of membrane damage compounds, was negatively related to chlorophyll stability (in Chinese bayberry; Hu et al. 2017), and TSS to maintain the water use efficiency at the cellular level was established with high regression coefficient (Kitayama et al. 2019).

In general, TSS in Catharanthus roseus and total carbohydrate in Hyoscyamus muticus grown under salt treatment was significantly lower compared to the degree of salt concentrations and developmental stages (Fatima et al. 2015; Abdelrazik et al. 2019). Regulation of TSS enrichment in leaf tissues (sucrose and fructose) of water spinach using NaCl elicitor has been previously confirmed, and it may play a key role in major osmotic adjustment when plants are subjected to salt treatment (Kitayama et al. 2019). However, the sugar content in NaCl-treated plants was reduced when PBZ was applied as a foliar spray. In strawberry fruit, total soluble solid (% Brix) was promoted by PBZ and it was still retained in plants subjected to a 5–10-mM NaCl elicitor (Jamalian et al. 2008, 2009). In addition, reducing sugars and water-soluble carbohydrates in root and flag leaf tissues were alleviated by increasing the PBZ application and salt concentration, thereby playing a role of major osmolyte to control cellular water potential (Hajihashemi et al. 2009).

AsA in the root and leaf tissues of Catharanthus roseus was regulated by NaCl elicitor and PBZ treatment to function as a non-enzymatic antioxidant (Jaleel et al. 2007). Moreover, a peak of AsA was observed in the strawberry fruit (58 mg per 100-ml juice) by mild NaCl and PBZ elicitors (Jamalian et al. 2008, 2009). Under salt treatment, AsA in tomatoes with overexpression of dehydroascorbate reductase (DHAR) genes was increased (Li et al. 2012). In addition, AsA in cotton was promoted by NaCl elicitor (Kumari et al. 2013). Alternatively, exogenous AsA foliar application is a way to increase endogenous AsA levels (Farouk 2011) and pistachio (Bastam et al. 2013). In water spinach, α-tocopherol was found to be enriched by NaCl elicitor (Kitayama et al. 2019). Tocopherol-deficient mutants (vte1 and vte4) of Arabidopsis showed a low endogenous α-tocopherol, leading to a salt-sensitive response (Ellouzi et al. 2013). Exogenous α-tocopherol application in soybean strongly improved biomass even exposed to NaCl conditions (Mostafa et al. 2015; Sereflioglu et al. 2017).

5 Conclusion

Plant height retardation and less NaCl toxicity in water spinach cv. WT using PBZ and NaCl elicitors were investigated. A low amount of Na+ in PBZ-treated plants under NaCl treatment was observed, leading to less toxicity in the root, stem, and leaf tissues of salt-treated water spinach. Photosynthetic pigment stabilization, PSII function, and net photosynthetic rate in PBZ-pretreated plants under NaCl treatment were stabilized using TSS as major osmolyte along with AsA, and tocopherol functioning as non-enzymatic antioxidants in PBZ-pretreated plants under salt treatment was demonstrated, which leads to better growth performance (Fig. 8). Moreover, enrichment of mineral nutrient and phytochemical compounds using PBZ and NaCl elicitors may further be implemented for large-scale production in the future.

Fig. 8
figure 8

A summary of evident morphological, physio-morphological, and biochemical traits of water spinach (Ipomoea aquatica) cvs. Chia Tai (CT, commercial cultivar; green stem) and wild type (WT cultivar, red stem) pretreated with 0, 17, and 51 M paclobutrazol (PBZ) for 7 days, and subsequently subjected to 100 mM NaCl for 7 days

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