Heavy metal toxicity induced by sewage water treatment in three different vegetables (lettuce, spinach and cabbage) was alleviated by brassinosteroid and silicon supplementation

2.1 Experiment site

The trial location was situated under semi-arid conditions, with mixed cultivation of orchards and field crops. The weather was characterized by extreme conditions i.e. hot summer and cold winter. Generally, temperatures begin to rise from mid-March and attain a maximum by May-June (mean maximum temperature is about 40 °C. The average rainfall in this area is about 96.2 mm, and the mean relative humidity is 61 %. The field trail was performed in the experimental zone of Horticulture Department, College of Agriculture, University of Sargodha in Punjab province of Pakistan.

2.2 Experiment

The field trial was conducted during October 2016–17. The seeds of 3 different vegetable species were collected from NARC, Islamabad, and sown on ridges. Seeds were dibbled manually by maintaining the proper distance. Three seeds of lettuce (Butter Head) and spinach (Local Sindhi) cabbage (Golden Acre) were sown per dibbled and thinned to a single plant after germination. All these vegetable varieties are mostly preferred in native region for home gardening. In addition, these varieties are high yielding capable to grow under stress conditions and have disease resistance.

Trials were conducted in split-plot two factorial designs where irrigational treatments were assigned in the main plot while vegetables were grown in micro plots. Theplot size was 90 cm × 300 cm and treatments were replicated thrice. Foliar application of Brassinosteroid @ 3.5 µM and Silicon @ 2 mM in case of lettuce and cabbage while Brassinosteroid @ 3.5 µM and Silicon @ 3 mM in case of spinach were applied at 15 days after germination in all three crops. The field was irrigated as per requirement with canal and sewage water according to the treatment plan.

2.3 Collection of data regarding growth parameters

For agronomic attributes, each seedling and its root were harvested after 35 days of plant growth, washed to clean its surface dirt, dried with tissue paper, and weighed by digital electric balance to determine the fresh weight. Harvested seedlings and roots were dried at 70 °C in an oven (KorlKolb 112 SL, Germany) for 48 h to constant mass. The dried weight of the seedling and root was determined through analytical balance. Area of leaf was estimated by using a digital meter (Ll-3000C) known as leaf area (LA) meter. The average leaf area was calculated by the method of (Binkley et al., 2002). The root length was measured with the help of measuring tape.

2.4 Collection of data regarding physiology and antioxidant enzymatic activities

Transpiration rate, Photosynthesis rate and stomatal conductance were determined by an instrument IRGA, CI-340 CID Biosciences, USA. All the gas exchange attributes were measured on top third leaf which was full expandedduring day hours. Water use efficiency (WUE) was measured by using following formula $$\mathit{WUE}=A/E$$ Where, A = Photosynthetic rate E = transpiration rate

Similarly SPAD meter (502 SPAD spectrum) was used to measure chlorophyll contents of leaf.

The Catalase (CAT) activity was examined by a procedure elaborated by Chance and Maehly (1955). While superoxide dismutase (SOD) was measured by the method of Giannopolitis and Ries (1977). Activity of Peroxidase (POD) enzyme from solution mixture was measured by spectrophotometer (Hitachi U-1800) at 470 nm wavelength. The activity of antioxidant enzymes was determined on the basis of protein contents. Activity of Ascorbate peroxidase (APX) enzyme was measured by theprocedure explicated by Mittler and Zilinskas (1991). It was based on the stoichiometric reduction of phosphor molybdenum by ascorbic acid.

2.5 Determination of silicon, cadmium, and lead concentration

Spectrophotometer was used to measure concentration of silicon in plant sample according to the method explained by Elliott and Snyder (1991). Whereas atomic absorption spectrophotometer (AAS) was used to determine the Cadmium (Cd) and lead (Pb) contents in plant samples.

2.6 Statistical analysis

A statistical analysis on collected datawas performed to compare treatment means by using LSD at a 5 % probability level. The software Origin 2023 (Origin Lab, Massachusetts, USA) was used to make graphs and find the Pearson correlation among various growth, physiological, and antioxidant enzymatic activities with silicon, lead, and cadmium concentrations.

3.1 Effect on growth attributes of leafy vegetables

Data (Table 1) regarding growth traits of lettuce, spinach, and cabbage showed that sewerage water irrigation reduced fresh weight of shoot, dry weight of shoot and leaf area as compared to canal water, as canal water contains less heavy metals than sewerage water. Mean while spraying BRs and Si on lettuce, cabbage, and spinach improved these parameters under the canal as well as sewage water irrigation. However, all these growth attributes were significantly (p ≤ 0.05) improved when lettuce, cabbage, and spinach were irrigated with canal water and sprayed with BRs and Si.

Data about the root components (Table 2) of leafy vegetables in terms of root fresh weight, root dry weight and root length had revealed the negative effects of sewage water irrigation on leafy vegetables owing to significant reduction in root attributes while roots of plants irrigated with canal water flourished vigorously. Foliar spray of BRs and Si under canal water irrigation showed promising (p ≤ 0.05) results by improving root fresh weight, root dry weight and root length while minimum values of root components were observed for plants irrigated with sewage water without BRs and Si application.

3.2 Effect on gas exchange parameters, water use efficiency and chlorophyll contents

The maximum and significant (p ≤ 0.05) increase in gas exchange attributes in terms of transpiration rate, stomatal conductance and photosynthetic rate (Table 3) and chlorophyll contents and water use efficiency (WUE) (Table 4) of leafy vegetables was observed under canal water irrigation along with leafy spray BRs and Si solutions. Whereas the lowest values were recorded when plants were irrigated with sewerage water without any treatment.

3.3 Effect on enzymatic activity

Data related to the activity of antioxidant enzymes such as SOD, POD, CAT and APX in lettuce, cabbage and spinach plants revealed (Figs. 1 and 2) that enzymatic activity was significantly (p ≤ 0.05) influenced byBRs and Si under both types of irrigation sources. BRs and Si mitigated the negative effects of metal stress and aggaravated the activity of antioxidant enzymes. The minimum enzymatic activity was recorded when plants were irrigated with sewerage water without BRs and Si whereas plants irrigated with canal water or sewerage water showed higher activity under the foliar spray of BRs and Si solutions.

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Fig. 1

Foliar effect of BRs and Si on superoxide dismutase (SOD) and catalase activity (CAT) of leafy vegetables irrigated with canal and sewage water. Means sharing same letter (s) do not differ significantly at p ≤ 0.05.

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Fig. 2

Foliar effect of BRs and Si on peroxidase dismutase (POD) and ascorbate peroxidase (APX) activity of leafy vegetables irrigated with canal and sewage water. Means sharing same letter (s) do not differ significantly at p ≤ 0.05.

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3.4 Effect on silicon, cadmium, and lead uptake

Data about concentration of Cd and Pb in vegetable plants showed (Fig. 3) increased content of Cd and Pb in plants were observed when sewerage water was applied as an irrigation source while minimum heavy metal (Cd and Pb) concentration was observed when irrigated with canal water. However, minimum Cd concentration was recorded in plants when irrigated with canal water along with a foliar spray of BRs and Si while plants irrigated with sewerage water and no foliar spray of BRs and Si showed maximum uptake of Cd and Pb concentration.

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Fig. 3

Foliar effect of BRs and Si on Si, Pb and Cd concentration of leafy vegetables irrigated with canal and sewage water. Means sharing same letter (s) do not differ significantly at p ≤ 0.05.

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The results regarding silicon contents in leafy vegetables showed (Fig. 4) that foliar spray of Si improved the Si contents significantly (p ≤ 0.05) and followed by BRs. The increase in Si concentration decreased the detrimental effects of Cd and Pb found in sewerage water. Minimum Si concentration was measured in plants when irrigated with sewerage water without foliar spray of Si. On the other hand, plants irrigated with canal water and foliar spray of silicon (Si) significantly improved Si contents.

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Fig. 4

Correlation between antioxidant enzymatic activities, growth parameters and metal concentration in lettuce. Superoxide dismutase (SOD), catalase (CAT) peroxidase (POD), Ascorbate peroxidase (APX), Si (silicon), Pb (lead), Cd (cadmium), SFW (shoot fresh weight), SDW (shoot dry weight), RL (root length) and Chl (chlorophyll contents).

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3.5 Pearson association between growth and physiological parameters, metal concentration, and antioxidant enzymatic activities

The data revealed that the shoot fresh and dry weight, chlorophyll content, and root length were significantly positively associated with POD, SOD, CAT, and APX in all the tested leafy vegetables (Figs. 4, 5, and 6). However, the Pb and Cd concentration was negatively related to the growth parameters and antioxidant enzymatic activities in spinach, cabbage, and lettuce. Silicon concentration was not significantly associated with shoot fresh and dry weight, root length, and chlorophyll contents but it was negatively associated with the concentration of Pd and Cd.

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Fig. 5

Correlation between antioxidant enzymatic activities, growth parameters and metal concentration in spinach. Superoxide dismutase (SOD), catalase (CAT) peroxidase (POD), Ascorbate peroxidase (APX), Si (silicon), Pb (lead), Cd (cadmium), SFW (shoot fresh weight), SDW (shoot dry weight), RL (root length) and Chl (chlorophyll contents).

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Fig. 6

Correlation between antioxidant enzymatic activities, growth parameters and metal concentration in cabbage. Superoxide dismutase (SOD), catalase (CAT) peroxidase (POD), Ascorbate peroxidase (APX), Si (silicon), Pb (lead), Cd (cadmium), SFW (shoot fresh weight), SDW (shoot dry weight), RL (root length) and Chl (chlorophyll contents).

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4.1 Growth attributes of vegetables

The stunted growth of all leafy vegetables was owing to the upregulation of heavy metals, particularly Cd and Pb under sewage water irrigation. The improvement in growth attributes in terms of fresh weight of shoot and roots and dry weight of shoot and roots, root length, and leaf area were attributed to the foliar spray of BRs and silicon. Foliar application of BRs accelerated cell division and cell elongation controlling the growth of plants under a heavy metal stress (Shafi et al., 2023). Likewise, Si foliar application also improved photosynthetic apparatus and nutrient absorption by confining the translocation of Cd an Pb which subsequently were associated with improved growth in plants (Hussain et al., 2019). Si spray also contributed to the intermodal elongation of shoots of leafy vegetables under metal contamination stress (Kabir et al., 2016).

4.2 Gas exchange parameters of vegetables and chlorophyll contents

Transpiration rate, Photosynthetic rate, stomatal conductance and chlorophyll contents were downregulated under sewage water irrigation due to the absorption of metal ions (Cd and Pb) in high concentration rather than other essential nutrients. Uptake of Cd and Pb in high concentration inhibited the exchange of CO₂ and O₂ in plant parts by increased chlorophyllase activity leading to disrupted stomatal conductance and transpiration rate which were imperative for the process of photosynthesis (Paunov et al., 2018; Hajihashemi et al., 2020). The upregulation in these gas exchange attributes was observed under BRs and Si application and they improved photosynthetic pigment by adjusting ion homeostasis and CO₂ fixation by reducing Cd and Pb ions and activity of chlorophyllase enzymes in plant tissues leading to encourage exchange parameters and chlorophyll contents in plant leaves (Adrees et al., 2015; Sonjaroon et al., 2018). BRs and Si foliar application to leafy vegetables prevented the accumulation of toxic elements and increased availability of essential ions in plants to improve photosynthetic rate by replacing central atom (Mg²⁺) of photosynthetic apparatus which is directly associated with increased stomatal conductance, transpiration rate and chlorophyll pigment (Agami, 2013; Shu et al., 2013).

4.3 Water use efficiency of vegetables

A decline in water use efficiency (WUE) by leafy vegetables was recorded under sewage water irrigation which is attributed to low relative water contents due to the toxic concentration of metal elements. Accumulation of Cd and Pb caused damage to plant plasma membrane due to the induction of reactive oxygen species (ROS) leading to a decrease in water transporters and subsequently low water use efficiency (Kaur et al., 2018). Foliar spray of BRs and Si reversed the loss of relative water contents by improving the membrane stability index owing to the limited supply of heavy metal ions. These also contributed to reducing oxidative damage to the plasma membrane by lowering toxic elements and upregulation of essential elements to increase membrane stability and relative water contents (Jan et al., 2018). Under stress environment foliar spray of BRs also increased osmolytes, particularly proline and Glycinebetain (GB) which improved water potential in plant tissues (Ahmad et al., 2015). All these processes are associated with improved WUE in leafy vegetables under both canal water and sewage water irrigation.

4.4 Activity of antioxidant enzymes in vegetables

The activity of antioxidants such as SOD, POD, CAT and APX in leafy vegetables decreased under sewage water irrigation while increased under canal water irrigation. Generally, the antioxidants activity increased under stress environment but our results are contradictory with the findings of other researchers such as (Fulekar et al., 2012) and (Garcia-Caparros et al., 2021). This might be due to the fact that in some situations protein misfolding happens under heavy metal stress that can decrease the activities of the enzymes. Moreover, for the activation of antioxidant enzymes, plants must be genetically capable of producing and activating antioxidant enzymes. Thus in some conditions, the plants are unable to activate the antioxidant system due to the lack of genetic capabilities (Tamás et al., 2014). Thus foliar application of BRs and Si is attributed to improve antioxidant enzymatic activities under both types of irrigation sources; canal water and sewage water. Under heavy metal stress, BRs and Si augmented their activity by lowering translocation of Cd metal through chelating agents (phytochelatins, phenols) and activating metal regulation through metal transporter within plants (Jan et al., 2018). The increase in activity of antioxidants caused a reduction in ROS species including hydrogen peroxide (H₂O₂) and singlet oxygen (O₂) and subsequently upregulated plant metabolic functions (Rahman et al., 2017).

4.5 Silicon, cadmium, and lead contents in vegetables

Increased concentration of silicon under stressed conditions is attributed to the adjustment of ion homeostasis by foliar application of Si and BRs in leafy vegetables (Wani et al., 2017). Furthermore, silicon application prompted to formation of silicates in cytoplasm leading to restricting the mobility of Cd and Pb in plants. Foliar application of Si also increased silicon contents by inducing silicon-containing compounds such as cysteine, glutathione, and methionine (Ji et al., 2017). Whereas transportation of Cd and Pb diminished due to increased absorption of other essential elements including P, K, Mg, Ca and Si, within plant tissues (Ahmad et al., 2018) through the antagonistic effect of nutrient ions. Thus depressing effect of Br and Si on Cd and Pb concentrations was mediated by ion homeostasis and the antagonistic effect of nutrient ions with each other (Waisi et al., 2017). Moreover, the induction of chelating agents like phenols and phytocelatins (PC) by BRs and Si provided favorable conditions for osmotic adjustment to vegetable plants under heavy metal stress (Cao et al., 2017).