Impact of lead on seed germination, seedling growth, chemical composition, and forage quality of different varieties of Sorghum

Due to heavy metal contamination, there are currently significant losses in agriculture and food security. Anthropogenic sources led to increased heavy metal contamination, which was becoming an attentive problem for agriculture and food safety. Among these metals, lead (Pb), which is toxic and affects crop production, has emerged as a standout due to its severe toxicity and relatively obscure mode of action.This study aims to compare the chemical composition and nutritional value of three tested sorghum cultivars grown in Pb-spiked soil with different levels of Pb (0, 100, 200, 400 and 800 mg kg−1). Also, the impact of different Pb levels on germination, fresh and dry weight of underground and aboveground parts, and morphological characters of three tested sorghum cultivars were assessed. The results emphasized that the germination index (GRI), radical and plumule length (cm), vigor index (VI), and tolerance index (TI) declined significantly by increasing Pb levels at all the examined cultivars. The morphological parameters of the examined cultivars decreased progressively with increasing Pb concentration for three tested sorghum cultivars. The nutritive values and forage quality of examined cultivars are negatively impacted by higher Pb levels, rendering them hazardous to animal consumption. These results may help scheme a mitigation strategy for lead injuriousness.

the chloroplasts' normal functioning by preventing the enzymes involved in chlorophyll biosynthesis, CO 2 fixation, and the pigment-protein complexation of photosystems [12]. Numerous HMs, including Pb, stimulate the higher production of Reactive oxygen species (ROS) in plant cells to a critical level, which then accelerates processes like lipid peroxidation, the proportion of saturated fatty acids versus unsaturated fatty acids (which increase in content) in cell membranes [13].
Numerous works have specified that trace elements will inhibit plant growth and crop production while enhancing the biosynthesis of several amino acids and carbohydrates [14][15][16]. Otherwise, reduction and inhibition of essential nutrients could occur due to HMs interfering with them [17,18]. The ability of plants to access HMs greatly depends on soil's chemical and physical properties and the plant species. Various plant species exhibit varying absorption capacity levels; some can take up high concentrations of HMs, while others can only take up one or a few metals. Moreover, plant tolerance could be varied depending on different plant growth stages [19]. The availability of HMs to plants is controlled by their chemical forms and the soil's total concentration. The plant will absorb high levels of HMs due to a low pH and high metal concentration in the soil [20].
Sorghum (Sorghum bicolor L.) is a multipurpose crop grown for industrial resolves, food, and animal feed. Sorghum is a C4 plant member of the Poaceae family and typically grows in hot, arid climates. Compared to other cereal crops, it is thought to be more resilient to various stresses, including drought, salinity, heat, and swamping [21][22][23].
Various works have been conducted on using sludge, wastewater, and solid waste, on the development and growth of Sorghum as well as the buildup of HMs in the soil and their transfer to different parts of sorghum [24,25]. Though, little work has examined the impact of these metals on nutrients that have not yet been obviously identified [26][27][28][29]. In this regard, the current work aims to evaluate the effects of different concentrations of Pb on germination and seedling growth, biochemical and mineral contents of three tested sorghum varieties.
Also, the impact of Pb on the forage quality of sorghum varieties.

Germination experiment
Seeds of different cultivars of Sorghum bicolor [red variety (S1), white variety (S2)& shahla variety (S3)] were identified and obtained from the Ministry of Environment water and Agriculture, K.S.A.
The experiment was set up in Petri dishes (90 mm) in the growth room of the research laboratory center. The filter paper was fitted into each sterilized Petri dish. Each Petri dish contained fifteen seeds. Five lead treatments were established using lead nitrate for irrigation purposes. Treatments to T0, T1, T2, T3, and T4 have 0, 100, 200, 400, and 800 mg of Pb/L. The arrangement of each Petri dish was a completely randomized design (C.R.D.). Ten ml of each treatment was added to the corresponding Petri dish. Germination was recognized when radicals emerged. The data of germination %, plumule, and radicle length was collected. The tolerance index and seedling vigor index were assessed according to Bewley and Black [30].

Pot experiment
At the greenhouse, an experiment was conducted between October and December 2020. The greenhouse temperatures and humidity levels were controlled to maintain a range of 20-28 °C and 50 ± 84 4%, respectively. Several types of Sorghum, grown for their bioenergy potential on Pb-contaminated soil, were put through an experiment to measure the amount the HMs could absorb. The soil was collected from nearby farmland and sands at a ratio of 2:1; then, it was air-dried, and sieved. The physicochemical characteristics of the studied soil are shown in Table 1. The electrical conductivity of this soil 88 was 0.44 dS cm -1 (E.C.), and the pH was 7.56. The total background level of Pb was 8.18 ± 2.1 mg 89 kg −1 , while the DTPA-Pb (available) background level was 2.100 ± 0.05 mg kg −1 . Pb was added to the soil as lead nitrate [Pb (NO 3 ) 2 ] at five different concentrations: 0, 100, 200, 400, and 800 mg Pb kg −1 dry soil. Based on the FAO/WHO [31] phytotoxicity threshold, the Pb levels of 400 and 800 mg kg -1 were chosen. After letting the treated soil equilibrate for around 30 days, a total of individual pots were employed in a completely randomized design (3 cultivars, 5 treatments and 6 replicate). Five seeds of each cultivar were put into 4 kg of soil in pots measuring 21 cm in diameter and 23 cm in height, and the plants were then grown in a greenhouse for 80 days under natural day/light conditions. After 15 days of seeding, we manually weeded the pots and irrigated them twice a week with a drip system, reducing the number of plants per pot from 5 to 3.

Soil analysis
Three soil samples were air-dried and sieved through a 2 mm sieve. Furthermore, 1/5 (w/v) water extract from the soil samples was prepared to determine pH with a pH meter (3510, Jenway, UK) and EC by a conductivity meter (Orion 150A+ , Thermo Electron Corporation, USA). Total Pb concentration was assessed according to Allen [32]; 1 g of soil was digested with a 20 ml tri-acid mixture of HNO 3 :H 2 SO 4 :HClO 4 (5:1:1, v/v/v) using an atomic absorption spectrophotometer (Shimadzu AA-6300; Shimadzu Co. Ltd., Japan). According to Norvell [33], the available Pb concentration of soil before cultivation was extracted and measured using DTPA (diethylene triamine pentaacetate).

Growth measurements
The crop plants under study were harvested 80 days after planting and dissected into roots and shoots. Each plant's root and shoot lengths were recorded. Additionally, the fresh and dry weights (g pot −1 ) were calculated by weighing the various components of each plant before and after oven-drying at 65 °C till constant weight.

Plant analysis
For each treatment, three samples of the collected plant material were collected, dried in the oven (at 65 °C), ground in a metal-free plastic mill until they were uniform in texture, and sieved through a 2 mm mesh sieve.

Chemical composition
For chemical analysis, the dried samples were ground into a fine powder. Nitrogen was estimated using modified Micro-Kjeldahl [34] and multiplied by 6 Forage Quality: The digestible crude protein (D.C.P. % in D.M.) was measured by using Eq. (2) according to Shaltout et al. [37].

Statistical analysis
Through the use of variance analysis, the obtained results were statistically examined using the S.P.S.S. Statistics 23 software. Each treatment's average and standard deviation for the chemical compositions of the soil and plants were calculated. Mean differences were compared using Duncan's test. The level for statistical significance was set at p ≤ 0.05 differences.

Impact of different levels of Pb on seed germination and seedling growth
The impact of different concentrations of Pb on three tested varieties of sorghum seed germination is presented in Fig. 1. Generally, seed germination is inhibited significantly (P ≤ 05) with increasing Pb levels ( Fig. 1). Examined sorghum varieties amended with 800 mg −1 of Pb, causing 27.59, 18.52 and 32.14%, inhibition of germination of seeds for sorghum S1, S2, and S3 respectively (Fig. 1). Lead ions may be interfering with seed enzymes associated with the hydrolysis and mobility of food reserves like carbohydrates and proteins necessary for embryo metabolism and development, which would explain the observed germination inhibition [42,43]. Furthermore, The inhibition effect of germination may be attributed to alterations in the selection permeability properties of cell membranes. Also, it could be due to loss of viability because of decreased energy generation by the embryo [43].
Germination may be obstructed due to Pb's antagonistic activity with the enzyme amylase and protease of higher plants, as suggested by Sengar et al. [48]. Additional investigation by Yang et al. [49] revealed that lead-induced stress resulted in boosting NADH-dependent synthesis of extracellular H 2 O 2 in the growing seeds, which may result in the arrest of wheat seed growth.
Reactive oxygen species (ROS) in cellular compartments, mitochondria, and chloroplast are formed as a result of excessive application of Pb [51].
Three tested varieties of Sorghum showed varying responses to Pb stress. Maximum tolerance was observed at low concentrations for Pb, and high metal contents led to decreased tolerance indices ( Table 2). From the results, it could be indicated that sorghum variety 3 had more tolerance than other tested sorghum varieties. Tolerance indices ranged from 39.43 to 79.59%, from 34.08 to 85.61%, and from 53.07 to 84.31%, for S1, S2, and S3, respectively ( Table 2). The low tolerance indices might have been due to the changes in the physiological mechanisms of the crop plants during their growth stages [52].
Exposure to high Pb levels leads to a reduction in root growth; it could be originated from abnormalities in cell division and/or cell elongation, which may explain this suppression of root growth [53,54]. Numerous mechanisms, including altered cell wall flexibility, suppression of microtubule growth and DNA synthesis, metal-induced chromosomal abnormalities, expansion of the mitotic cycle, and decreased glutathione pool, are linked to reduced cell division and elongation rates in plant roots [55].
Moreover, Srinivasan et al. [56] reported growth inhibition is a typical response to heavy metal stress and is also one of the essential agricultural indices of heavy metal tolerance.

Impact of Pb-treatments on the morphological characters
Data regarding morphological features revealed that Pb treatments significantly affected the root and shoot length, no. of leaves, and plant biomass (fresh weight, dry weight) of the three tested sorghum varieties (Table 3). Plant morphological criteria are a significant indicator of characterizing the growth performance of plants after being subjected to HMs. The plant appeared to exhibit symptoms of growth inhibition when exposed to high levels of Pb toxicity. High doses of lead exposure can be restricted by plant biomass.The highest decline recorded was in root dry weight was decreased by 73, 75, 71%, and shoot dry weight by 80, 65, and 59% of sorghum S1, S2, and S3, respectively, at Pb 800 mg Kg −1 as compared with control (Table 3). This finding with in line with Kanwal et al. [46] on wheat, Ramana et al. [57] on cotton, Alaboudi et al. [58] on sunflower, and Bassegio et al. [59], who reported that the highest dry weight of B. juncea was recorded in control and then declined by increasing Pb content in the soil, indicating the negative effects of Pb on plant growth. The major reason behind plants biomass reduction is a reduction in the rate of photosynthesis and nitrogen metabolism [60].
Lead toxicity has morphological impacts that include elongated, inclined, and stumpy distended roots, numerous secondary roots per unit root length, and a stoppage of root elongation [61,62]. After being exposed to lead for 48-72 h, the root cells of Allium sativum showed signs of mitochondrial dilatation, abortive cristae, vacuoles in the endoplasmic reticulum and dictyosomes, fractured cellular membranes, and hyperpycnotic nuclei [63]. Increased Pb exposure was proven to suppress plant biomass [64,65].
Plant development and growth could be impacted because the HMs could obstruct the plant metabolism due to interactions with enzymes and biochemical reactions in the plant tissues [66].

Impact of Pb-levels on nutritional value and fodder quality
The nutrient values of sorghum cultivars were studied in terms of parameters viz. protein content, ether extract, ash, fiber, nitrogen-free extract, detergent fiber (ADF), and neutral detergent fiber (NDF). A linear significant (p ≤ 0.05) decrease in the protein contents was noted with an increase in Pb concentration. Maximum reduction was detected in those plants that grew in soil spiked with 800 mg Pb kg −1 (Table 4). In this concern, Kanwal et al. [46] found that the protein content decreased by about 81% in a wheat plant grown in soil amended with 800 mg kg −1 of Pb compared with the control. Total protein value declined in the plant due to increased tension. This decline in plant proteins may enhance the contribution of the fiber materials in the plant [67,68]. Also, It might be due to less nitrogen absorption by the plant under heavy metal stress like Pb toxicity, as nitrogen is utilized directly in protein synthesis by the plant. Hence, nitrogen absorption decreased, and Pb accumulation increased in the plant, resulting in reduced crude protein content and decreased overall plant growth in terms of sugars, proteins, and yield [69].
In the present study, the content of ether extract, fiber, ADF, and NDF increased significantly by increasing Pb concentration for three tested sorghum varieties ( Table 4). The data revealed that the ADF and NDF content increased significantly by (mean from the three tested sorghum varieties) 1.37 and 1.14 times grown at soil amended with at 800 mg Kg −1 as compared to control toxicity (Table 4). In the current work, the enhancement in ADF and NDF values in the examined sorghum cultivars under Pb stress might be due to increased lignification and silicification as a defense mechanism against harsh circumstances in plant cells, which in turn give the cell wall firmness and rigidity and protects the cell from an osmotic burst. It resulted in the increased fiber components of the cell wall, which makes the plants less digestible for animals [70][71][72][73].
The nutritional values of the forage crops are the outcome of the two major components: (i) nutritive value and (ii) voluntary intake by grazing animals (livestock) and palatability [74,75]. In this experiment, the nutritional value of three tested sorghum varieties grown in soil amended with different concentrations of Pb was assessed according to calculated equations.
Total digestible nutrients (TDN) and gross energy (GE) consider significant indicators for forage quality assessment. The values of both TDN and GE content were increased significantly (P ≥ 0.05) by increasing Pb contents (Table 5). On the other hand, the results of Gross energy (GE), Digestible energy (DE), Metabolized energy (ME), and Net energy (NE), showed a non-significant effect of Pb contents on their values. Meanwhile, The digestible crude protein (DCP) was decreased significantly by increasing Pb contents. The same finding is linear with [76].

Conclusion
Sorghum has significant economic importance; it is multipurpose and used as food and a fodder crop worldwide. In areas that are susceptible to drought, it might be employed as a viable approach to food supplies. The results from the current investigation suggest that Pb has adverse effects. This is supported by the impact of Pb toxicity on seed germination, seedling growth, morphological characters, chemical composition, and nutritional values of examined cultivars of Sorghum, which led to direct and indirectly affect on livestock health. In the current investigation, the application of Pb caused a significant decline in plant growth, chemical composition, and nutritional quality compared to the control. To restore nutritional quality and plant growth, appropriate strategies must be employed to overcome Pb toxicity in crop plants. These results may help scheme a mitigation strategy for lead injuriousness.
Author contributions HO and RF methodology: HO and RF analysis: HO and RF data curation: HO writing-original draft preparation: HO writing-review and editing HO, and RF. All authors have read and agreed to the published version of the manuscript.
Funding Each author declares that no funds, grants, or other support were received during the preparation of this manuscript.
Availability of data and materials Not applicable.

Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
Ethical approval Not applicable.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.