Role of polyethylene glycol to alleviate lead stress in Raphanus sativus

Seed priming and growth conditions

The seeds of radish (Variety: Mino Early syn. Diakon, White), a fast-growing Japanese cultivar commonly cultivated in many areas of Pakistan due to its non-bolting, heavy-yielding nature were obtained from RSC (Roshan Seed Corporation), Lahore, Pakistan. Firstly, the seeds were surface sterilized with 1% sodium hypochlorite for 10 min and air dried in an ambient environment after washing with distilled water thrice. Lead acetate (Pb (C₂H₃O₂)₂ as a source of Pb and polyethylene glycol (PEG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The seeds were primed in varying concentrations of PEG solutions in four beakers in which 100 ml of 10%, 20%, 30% and 40% PEG were taken and 30 seeds in each beaker were placed for 16 h, under low light exposure at 25 °C. After priming seeds were washed thrice with distilled water and dried under shade for 12 h (Shatpathy et al., 2018). Non-primed seeds were considered as control. A pot experiment was performed in a wire-house, Botanical Garden, Institute of Botany, Quid-e-Azam campus, University of the Punjab, Lahore (N 31°30′4.3236″, E 74°18′ 5.4684″). During this study, there was 18 ± 3 °C/12 ± 2 °C (day/night), 56–68% relative humidity, and 11 h photoperiod. Loam soil obtained from 0–12″land depth of the Botanical Garden, the University of Punjab, composed of 139, 510, 284 and 20 g/kg sand, clay, silt and organic matter, respectively, at 6.8 pH was used for the current study. Moisture concentration of soil at field capacity was 72% and its electrical conductivity was 0.19 dS m⁻¹. While, it had 9.6 mg P, 2.39 g Si, 2.87 mg Ca, 1.65 mg K, 5.07 g N and 5.37 mg Mg in kg⁻¹. Soil was contaminated with 75 mg/kg Pb and kept for 15 days before sowing of radish seeds. Afterward, 2 kg of Pb-spiked-soil and Pb-free soil were filled in 10″ plastic pots. A total of thirty pots were used with CRD. Then five primed and non-primed radish seeds were sown in the allocated pot and each treatment group was replicated three times to ensure accuracy in results. Thinning was performed after 15 days and three seedlings were allowed to grow further in each pot. Each pot was supplemented with 100 mL 50% Hoagland’s solution on each seventh day.

Germination test

Over 10-days period, the primed seeds underwent germination defined by the emergence of a two mm long radical (Kaya et al., 2006). The germination process was meticulously monitored with observations recorded at 24-hour intervals. Each treatment underwent three replications to ensure precision in statistical analysis. The 20-day-old seedlings were systematically examined to assess various growth and germination characteristics, including overall germination percentage and seedling vigor index. Furthermore, an extensive range of parameters about growth, nutritional content and physiological traits underwent comprehensive analysis, conducted 60 days following the germination initiation. This approach was employed to ensure a thorough evaluation of the seed’s developmental progression.

The germination assessment procedures outlined in the authoritative manual of the Seed Analysis Association (Scott, Jones & Williams, 1984) were implemented to ascertain the number of viable seeds that successfully underwent germination. The quantification of germinated seeds was executed on the 9th day following seed deployment and the germination percentage was determined using the established conventional methodology (Shakirova et al., 2003) as follows: ${\displaystyle \text{Germination Percentage (GP)}=\frac{N1}{N2}\times 100}$ Here, N1 is the number of germinated seeds, while N2 is the total number of seeds sown.

Seedling vigor index (SV1)

The computation of the SVI was executed by employing the formula specified by the Association of Official Seed Analysis (1990) as delineated below: ${\displaystyle \text{SVI}=\left(\text{Root length}+\text{Shoot length}\right)\times GP.}$

Assessment of morphological parameters

Following 60 days of germination, morphological parameters including plant shoot length and plant root length from three randomly selected plants of each treatment were evaluated, while pod and seed counts were done after 90 days of germination.

Determination of fresh and dry weight of plants

The fresh shoot weight of three randomly selected seedlings (60 days following germination) from each treatment was analyzed by electric weighing balance. Before the weighing process, any surplus moisture present on the surfaces of the plants was eliminated using tissue paper. The mean values were presented as the total weight of plants in their fresh state. Subsequently, the plants were subjected to a controlled drying process within a thermal chamber set at 60 °C for 48 h. Once the drying process reaches a point where there is no discernible alteration in the weight of the plants, the dry weight is duly documented and recorded. Subsequently, the average value for all plant specimens was then computed.

Estimation of gas exchange parameters

IRGA (Li-COR Inc, Lincoln, NE, Biosci., USA) was used to examine gas exchange properties such as net photosynthesis rate transpiration rate and stomatal conductivity, from fully stretched leaves at 10 a.m.

Determination of chlorophyll and carotenoid pigments

Using a spectrophotometer set to measure at 440, 645 and 663 nm, the amounts of chlorophyll a, chlorophyll b and total chlorophyll were determined according to Khaleghi et al. (2012). Fresh plant leaves were crushed and mixed with 80% acetone. The test tube extract was then transferred to the cuvette. Carotenoid content was estimated by using the Lichtenthaler & Wellburn (1983) approach as follows: ${\displaystyle C_{\mathrm{x+c}}=\left(1000A_{470}-2.27C_a-81.4C_b\right)/227}$ where: C_a = chlorophyll a content, C_b = chlorophyll b content and C_x+c = carotenoids content

Evaluation of plant mineral status

The plant samples (400 mg) were subjected to digestion using aqua regia at a 3: 1: 1 ratio (HNO₃: HCLO₄: H₂SO₄, V/V). After cooling, the digested components were filtered through filter paper and a final volume of 100 mL was achieved by adding distilled water. The quantification of mineral content in plant samples was determined using atomic absorption spectroscopy (nitrogen) and a flame photometer (phosphorus, potassium).

Estimation of proline

The extraction and evaluation of free proline in radish plants were conducted following the acid ninhydrin method described by Bates, Waldren & Teare (1973). For this purpose, 100 mg frozen leaves were homogenized with two mL of sulphosalicylic acid. The samples were agitated at a rotational speed of 750 revolutions per minute (rpm) for 30 min. A 0.8 mL volume of supernatant was mixed with an equal volume of acid-ninhydrin reagent. The resultant mixture underwent vigorous agitation and was subsequently allowed to equilibrate at ambient temperature for 25 min, during which phase separation occurred. The absorbance of toluene was measured at a wavelength of 520 nm with pure toluene serving as the reference.

Assessment of accumulation coefficient and metal tolerance index

Aqua regia an acidic mixture was used to decompose the desiccated plant material. The resulting mixture was filtered and diluted with distilled H₂O to reach a volume of 100 mL. The concentration of Pb in both root and shoot tissues was quantified using an atomic absorption spectrophotometer. The accumulation coefficient was calculated using the following formula proposed by Al-Farraj et al. (2010): ${\displaystyle Accumulationcoefficient=\frac{Concenteration\left(ShootorRoot\right)}{Concenterationofsoil}.}$

To calculate the metal tolerance index (MTI), the following equation was used: ${\displaystyle \text{\%}\text{MTI}=\frac{\text{Dry weight of treated plants}}{\text{Dry weight of untreated plants}}\times 100.}$

Estimation of total soluble proteins and antioxidant enzymes

The present study employed cryogenic grinding with liquid nitrogen to process fresh plant material, thereby enabling subsequent analysis of protein content and enzymatic activities. After cryogenic grinding, the resulting finely ground plant material was immersed into a three mL phosphate buffer solution. Subsequently, the sample underwent centrifugation at 14,000 rpm for 30 min at 4 °C. The resultant supernatant from the centrifuged mixture was meticulously collected and subsequently stored at 0 °C. This strategic storage condition was implemented to facilitate the subsequent quantitative assessment of protein content, as well as enzymatic activities of POD, CAT and SOD.

The Biuret technique developed by Racusen & Johnstone (1961) was employed to estimate soluble protein concentration. To initiate the reaction, two mL of Biuret reagent was mixed with two mL of the supernatant. Subsequently, the optical density at 545 nm was measured using a spectrophotometer. The quantification of protein concentration was achieved by referencing a standard curve consisting of pre-established protein values as follows: ${\displaystyle \text{Protein contents}\left(\frac{mg}{g}\right)=\frac{\text{Curve value}\times \text{Total extract}}{\text{Extract used}\times \text{Weight of tissue used}}\times 1,000.}$

The approach established by Luck (1974) was used to evaluate POD activity. The experimental setup involved a buffer solution, guaiacol, enzyme extract and hydrogen peroxide solution. The quantification of POD was achieved by ascertaining the duration necessary to attain a 0.1-unit increment in absorbance at 240 nm. ${\displaystyle \text{Peroxid aseactivity}\left(\frac{mg}{g}\right)=\frac{\text{Absorbance}\times df}{\text{Weight of tissue used}\times EU}\times 1000.}$ df = dilution factor EU = extraction unit

The CAT activity assessment was conducted using the Beers & Sizer (1952) technique, in the presence of two buffer solutions. For this purpose, a reaction mixture comprising 2.9 mL of buffer B and 0.2 mL of enzyme extract was used. In contrast, the control group contained just three mL of buffer A. The duration required for the reduction of absorbance (at a wavelength of 240 nm) from 0.45 to 0.40 was utilized for CAT activity evaluation. This activity was subsequently quantified as units per mL of the enzyme. This method ensures a rigorous and standardized approach, contributing to the robustness and reliability of the scientific investigation. ${\displaystyle \text{Catalase activity}\left(\frac{\text{units}}{\text{mLenzyme}}\right)=\frac{3.45\times df}{Min\times 0.1}.}$

Maral, Puget & Michelson (1977) conducted a study wherein they employed spectrophotometric analysis to measure the activity of SOD. This was achieved by evaluating its capacity to hinder the photochemical reduction of nitroblue tetrazolium. For this purpose, two test tubes containing a mixture of sodium cyanide, NBT methionine, riboflavin and EDTA as a substrate were employed in the technique. The absorbance of the illuminated tube at a wavelength of 560 nm was compared to that of the non-illuminated mixture. The measurement of SOD activity was conducted in mg of protein. ${\displaystyle \text{\%}\text{Inhibition}=\frac{\text{absorption of control sample}-\text{absorption of experimental sample}}{\text{absorption of experimental sample}}\times 100.}$

Determination of hydrogen peroxide and malondialdehyde content

Hydrogen peroxide extraction from plant tissues was induced following the methodology proposed by Patterson, MacRae & Ferguson (1984). The pulverization of fresh leaves was conducted, in conjunction with the utilizing TCA (trichloroacetic acid) and activated charcoal. The sample underwent centrifugation at 3,000 rpm for 20 min at a temperature of 4 °C. The filtrate was then divided into one mL aliquots. One of these containers was filled with 81 g of CAT and kept at ambient temperature for 10 min. A one mL volume of colorimetric reagent was introduced to both the aliquots and allowed to incubate for 10 min at a temperature of 30 °C. The spectrophotometric measurement was conducted to determine the absorbance at 505 nm.

1g of foliage was subjected to vortex with the solutions of thiobarbituric acid (six mL) and trichloroacetic acid and for 15 min underwent incubation at 95 °C. Subsequently, the mixture was cooled and then centrifuged (10,000 × g) for 20 min, which finally led the supernatant to a spectrophotometer for quantification of malondialdehyde (MDA) content (Zheng et al., 2012).

Statistical analysis

The data obtained represents the mean value of three replicates. RStudio software was employed to conduct principal component analysis (PCA) and determine Pearson correlation coefficients among the observed variables. SPSS version 20 was used to calculate the analysis of variance. Subsequently, Tukey’s Honestly Significant Difference (HSD) test was employed to assess the treatment means, enabling the ranking and comparison of significantly distinct treatments at a probability level of P < 0.05.

Seed germination and seedling vigor index

An observable increase in the germination % and seedling vigor index (SVI) has been noted due to the Pb and PEG applications. The absence of the PEG application caused a significant decline in the germination % (36.6%) and the SVI (59.13%) was noted under Pb stress conditions compared to control plants. Table 1 presents the observed fluctuations in germination percentage and morphological parameters in response to the imposition of Pb-induced stress. The seedlings subjected to Pb-induced stress exhibited a reduced germination percentage (47.7%) compared to plants in the control group. Values for maximum growth performance (GP) and SVI were recorded at the PEG-30% treatment, exhibiting elevated germination compared to both the control group and Pb-contaminated treatment, as well as other PEG treatments. The germination rate in the PEG-30% treatment showed a notable increase of 19.04% compared to the control. Under the influence of Pb stress, reduction is observed in various growth attributes compared to the control group. Specifically, there is a reduction of shoot length (48.83%) and root length (32.89%) under Pb stress. The recorded data under Pb stress conditions revealed that shoot and root length significantly increased in the presence of PEG-30%. Specifically, a remarkable increase of 62.79% in shoot length and 50.98% in root length was observed compared to the conditions solely subjected to Pb stress.

Biomass assessment

The radish plants subjected to Pb-induced stress showed a notable decline in the fresh weights of both their shoot and root, with a reduction of approximately 32.89% and 35.48%, respectively, compared to the control seedlings. The data exhibited that the PEG-primed seeds revealed a distinct positive impact on fresh and dry biomass production, in contrast to plants subjected to Pb-induced stress. Lead stress reduced shoot and root biomass production by 60% and 50%, respectively, in seedlings. Growth parameters increased with increased PEG concentrations. Nevertheless, it is important to mention that an increase in the concentration of PEG (up to 40%) resulted in a slight decrease in growth. This decline, though relatively minor, was still comparable to the control group and showed no significant difference when compared to plants subjected to Pb-stress. The application of PEG-30% significantly enhanced the fresh weight of the shoot (54.54%) and root (40%) compared to plants that did not undergo priming and were exposed to Pb-stress. The seed that underwent priming with PEG-30% showed a notable reduction in fresh and dry biomass production compared to the control group (Table 1). Maximum pods and highest seed number per pod in treated plants were counted in PEG-30%+Pb treated plants, which were 7.29% and 30% higher, respectively, compared with the plants grown in a stress-free condition (control). Pb toxicity resulted in a notable decrease in the quantity of pods and the number of seeds per pod. The observed pod count per plant in Pb stress conditions exhibited a 65.62% reduction compared to the number of pods per plant in the control group. Additionally, the number of seeds per pod in plants treated with Pb was 70% lower than that of the control group (Table 1).

Estimation of gas exchange parameters

Plants subjected to Pb stress exhibited a notable reduction in gas exchange traits, including net photosynthetic activity (A), transpiration rate (Ei) and stomatal conductivity (gs), as compared to the control group. Specifically, the observed decreases were 46.15%, 36.36%and 49–25% for stomatal conductivity, transpiration rate and net photosynthetic rate, respectively, compared to the control plants. Polyethylene glycol (PEG) primed seeds exhibited enhanced gas exchange parameters, thereby mitigating the unfavorable effects of Pb in radish. Specifically, when exposed to a combination of PEG-30% and Pb, these plants demonstrated an 85.71% increase in stomatal conductivity, 57.14% enhancement in transpiration and a 93.59% increase in net photosynthetic rate in contrast, plants that were solely treated with Pb did not exhibit such improvements (Fig. 1).

Determination of photosynthetic pigments

The radish plants exhibit an apparent decline in their photosynthetic pigments when subjected to soil contaminated with Pb. Compared to control, Pb reduced chlorophyll b synthesis (0.2 mg/g FW) more than chlorophyll a (0.4 mg/g FW). Our results showed a decrease of 45.94% in carotenoids, 56.25% in total chlorophyll and 60% in chlorophyll b content. Significant enrichments were noted in the levels of chlorophyll content and carotenoids within the plant that underwent pre-treatment with PEG, under both Pb-stressed and non-stressed soil conditions.

The application of PEG has been found to enhance the synthesis of photosynthetic content. In particular, the best results were obtained by seed priming with PEG at a concentration of 30%. This concentration increased Chl a by 60%, Chl b by 61%, total chlorophyll by 56.25% and carotenoids by 58.33% under Pb-induced stress (Table 2).

Evaluation of plant mineral status

Table 3 showed data on nitrogen (N), phosphorus (P) and potassium (K) uptake found in the leaf tissues of R. sativus plants. Plants subjected to Pb treatment exhibited a reduction in their N, P and K levels. It was noted that the N content decreased by 43.47%, the P content by 28.96%and the K content by 30.35% compared to the control plants. Leaf samples exhibited significantly elevated N, P and K levels in plants subjected to PEG-30%+Pb treatment over stress plants. PEG-30%+Pb upsurges the N by 50.93%, P by 46.4% and K by 38.18% over Pb-induced seedlings.

Assessment of Pb uptake

Table 4 indicated that plants grown in Pb-enriched soil showed enhanced Pb uptake by 3.9 µg/g DW in plant roots and 0.12 µg/g DW in plant shoots. Nevertheless, PEG treatment inhibited Pb translocation and uptake in both roots and shoots. Pb concentrations in roots grown under 10, 20, 30and 40% PEG were 3.48, 2.40, 3.77 and 2.73 µg/g DW and in PEG-treated plant shoots, they were 0.336, 0.493, 0.246 and 0.166 µg/g DW. PEG 20 and 30% reduced the Pb uptake by 50% and 21% in roots and 16.86% and 53.51% in shoots, respectively.

Assessment of metal tolerance index and accumulation coefficient

The plants cultivated in soil contaminated with Pb exhibited AC of 0.05 and MTI of 42.33%. Application of 20% PEG and 30% PEG resulted in significantly increased MTI values by 66.32% and 58.77%, respectively compared to plants affected by Pb contamination. The seed primed with 20% PEG under Pb stress, resulted in a significant reduction of the apparent conductivity (AC) value by 40% (Table 4).

Estimation of antioxidant enzyme activities

The current investigation explains the physiological reactions of antioxidant enzymes in the presence of Pb and PEG treatments. According to studies, the utilization of various concentrations of PEG (10%, 20%, 30% and 40%) in isolation, as well as in combination with Pb, exhibited a notable enhancement in the superoxide dismutase (SOD) activity of the radish plant when compared to the control group. Similarly, the levels of catalase (CAT), hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and peroxidase (POD) activities were found to be elevated. The comparative investigation among plants treated with Pb and control plants demonstrated that the Pb significantly upsurge the levels of SOD, POD, CAT, H₂O₂ and MDA by 86.08%, 44.07%, 54.47%, 81.48% and 54.71% respectively (Fig. 2). The Pb+ PEG-20% treatment revealed the most elevated SOD activity. This treatment demonstrated a notable increase of 86.50% compared to the control group and a slight increment of 0.22% compared to plants treated solely with Pb. The Pb+PEG-40% treatment exhibited the highest level of POD activity, surpassing that of plants treated with Pb by 2.91% and the control by 48.34%. The plants subjected to the combination of Pb+PEG-30% showed the most significant elevation in CAT activity (55.26%), over the plants exposed solely to Pb stress. Conversely, the plants treated exclusively with Pb demonstrated the highest levels of H₂O₂ and MDA contents showing a considerable 81.48% and 54.71% increase respectively compared to the control treatment.

Estimation of proline content

When PEG was applied as a seed pre-treatment, proline concentration in radish plants under Pb stress conditions significantly increased. Under Pb-stress, PEG-30% showed the highest proline concentration, measuring about 5.24 mg/g. The findings of this experiment indicate that the proline concentration of radish plants was significantly higher in the treatment group when different PEG concentrations (10%, 20%, 30% and 40%) were used alone or in combination with Pb. Upon subjecting plants solely to Pb, a substantial and prominent increase in proline content of 43.28% was observed compared to the control group (Fig. 3).

Estimation of soluble protein content

The presence of Pb hinders the plant’s ability to synthesize proteins, leading to a subsequent decrease in various growth parameters. Upon comparing plants subjected solely to Pb treatment with the control group, a notable and significant reduction of 60% in their protein content was observed. Application of pre-treatment of PEG exhibited a distinct positive impact on the protein concentrations in radish plants subjected to Pb stress conditions. PEG-30% demonstrates the highest protein concentration by 1.3 mg/g under Pb-induced stress. This concentration closely resembles the control values, indicating the Pb mitigating effects of PEG (Fig. 3).

Correlation between various growth and physio-biochemical attributes with Pb uptake and accumulation

A Pearson’s correlation analysis was employed to identify the association between the growth and physio-biochemical characteristics of PEG-treated R. sativus cultivated in soil contaminated with Pb (Fig. 4). The concentration of Pb in the shoot exhibited a positive correlation with the accumulation coefficient in the shoot and the proline content in plants of the species R. sativus. In contrast, the concentration of Pb in the shoot exhibited a negative correlation with various factors including metal tolerance index, potassium, root dry weight, total phenol content, stomatal conductance, germination percentage, total chlorophyll, transpiration rate, shoot dry weight, photosynthetic rate, protein content, fresh weight of root and shoot, root length and carotenoids. According to these findings, PEG showed efficacy in reducing the negative impacts of Pb stress by enhancing the absorption of vital nutrients and modifying antioxidant enzymes.

Principal component analysis

The cultivation of R. sativus on soil contaminated with Pb, supplemented with PEG, exhibited a correlation between growth and various physiological and biochemical traits. This relationship was elucidated through the utilization of loading plots derived from principal component analysis, as depicted in Fig. 5. The initial two principal components, Dim1 and Dim2, constitute the predominant proportion of all components and encompass over 79.0% of the entire dataset. Dimension 1 constitutes 57.2% of the dataset, while Dimension 2 accounts for 21.8% of the dataset. A positive correlation was observed among various variables, including potassium, magnesium, sodium, zinc, total phenol content and stomatal conductance. The proline content, metal tolerance index and accumulation coefficient exhibited a significant negative correlation. The largest impact on the development and antioxidant capacity of R. sativus was seen under Pb stress. Plant growth was negatively correlated with it, but antioxidant capacity was positively correlated. PEG primarily increased plant height, aboveground biomass and POD, CAT and SOD activity to boost plant growth and antioxidant capacity (Fig. 5).