In the current study, red clover plants planted in Pb-polluted soil had worse growth, yield, and physiological performance than plants cultivated in unpolluted soil. As the soil Pb contents increased, there was a notable reduction in growth, yield, and physiological performance. These results aligned with the research conducted by Zeng, et al. 34, Saleem, et al. 35 and Shah, et al. 36. The biomass reduction and growth inhibition induced by Pb exposure may be attributed to the inhibition of nutrient absorption by Pb, which disrupts the permeability of the cell membrane and damages plants photosynthetic activity 4. Plant cells may produce a variety of reactive oxygen species (ROS) in response to elevated Pb concentrations in the soil. This surge in ROS leads to severe oxidative damage, adversely affecting plant growth 37. In summary, high Pb levels in soil often cause plant poisoning as Pb accumulates in plant tissues, limiting plant growth and reducing yield.
However, a number of studies have demonstrated that applying biochar can improve plant growth, physiological performance, and yield while reducing the harmful impacts of heavy metals 38–40. On the one hand, corn straw biochar has relatively high concentrations of trace elements and inorganic nutrients, improves soil fertility, and provides the necessary elements (Na, P, K, Ca, Mg, etc.) for plant growth 41. On the other hand, the surface of biochar possesses various functional groups that boost its cation exchange capacity. These groups can be substituted by heavy metals, thereby decreasing damage to plants from these heavy metals 42. Our results showed that adding biochar to Pb-contaminated soil reversed the toxic effects of Pb, leading to improved growth, physiological performance, and yield in red clover compared to plant cultivated in Pb- polluted soil without adding biochar. It is possible that the transfer and fixation of Pb in biochar reduced the migration of Pb to plant roots and shoots, thus reducing Pb toxicity and increasing plant development. In addition, the auxiliary effect of biochar varies with the dose of biochar 43. Ma, et al. 44 found that 2% corn stalk biochar application significantly decreased Pb accumulation in various tissues of tomato plants, thus promoting their growth. In this study, 5% biochar addition had the greatest pronounced impact on plant height, root length, and biomass of red clover.
Excessive heavy metals in plant tissues can impair plant physiology, primarily by decreasing photosynthetic activity 45. Chlorophyll a, which serves as the primary pigment for light capture, functions as the reaction centre for photosynthesis in leaves and plays a pivotal role in this process. Chlorophyll b acts as an accessory pigment with a photoprotective effect, aiding chlorophyll a in the photosynthetic process within leaves 46. Additionally, carotenoids, auxiliary pigments that aid in light harvesting, play a crucial role in photoprotection through nonphotochemical quenching of excess light energy, safeguarding the plant's photosynthetic system 47. According to a study conducted by Nelissen, et al. 48, biochar can increase the chlorophyll contents by promoting nitrogen mineralization, increasing the amount of nitrogen available for absorption by plants. This study showed that when biochar was added, particularly at a 5% dose, significantly elevated the levels of carotenoids, chlorophyll a and chlorophyll b in red clover leaves exposed to Pb. This improvement might be attributed to the ability of biochar to mitigate heavy metal toxicity through the immobilizing heavy metals and enhancing nutrient uptake by plants. This helps to maintain chloroplast structural stability, safeguards photosynthetic pigments, boosts photosynthesis, and enhances the growth of red clover.
Under heavy metal stress, the ROS content increases sharply, which causes lipid peroxidation in cell membranes, damaging nucleic acids, lipids and proteins in plant cells and ultimately inhibiting plant growth and yield if these substances cannot be removed in time 49. However, plants can enhance antioxidant defence mechanisms through upregulating crucial antioxidant enzymes activities, which play vital roles in the detoxification of ROS 50,51. SOD converts O2− into H2O2 and O2, acting as the first line of defence in the reactive oxygen scavenging and plant antioxidant enzyme systems 52. Subsequently, H2O2 is broken down by CAT and POD into H2O and O2, further preventing ROS damage in plant 53. Our study found that incorporating biochar significantly boosted activities of antioxidant enzymes in red clover grown in Pb-contaminated soil, indicating an enhanced defence response to oxidative stress. In particular, the 5% biochar treatment had better effects than did all the other treatments in improving SOD and POD activities. Numerous studies have demonstrated that by stimulating the antioxidant enzymes, biochar can strengthen the antioxidant defence 54–56. In addition to ROS scavenging, osmotic regulation is also necessary for plants growth 57. Proline regulates the osmotic pressure in plants by acting as a free radical scavenger, metal chelator, and protein stabilize 58. In this study, when plants were exposed to Pb stress, large quantities of proline was produced, which is in line with the results of Mu et al Mu, et al. 59. After the addition of biochar, there was a noticeable reduction in proline accumulation, which could be explained by the ability of biochar to decrease heavy metal toxicity to plant leaves and decrease cellular damage. As a result, the proline content decreased, indicating the mitigation of stress-induced responses in red clover.
Our study suggested that the addition of biochar substantially decreased the Pb content in shoots and roots compared to that of plants cultivated in biochar-free Pb-contaminated soil, mirroring the findings presented by Wang, et al. 40. The addition of biochar to Pb-polluted soils resulted in a decrease in Pb contents in the roots and shoots, possibly because the addition of biochar increased the soil pH, which is crucial for the dissolution and absorption of heavy metal 60. When biochar with high pH is used to remediate soils, the oxygen-containing functional groups and mineral components on the surface of biochar react with heavy metals via complex precipitation, which fixes and passivates heavy metals, thus reducing the bioavailability of heavy metals in soil as well as their absorption by plants 61,62. In this study, the addition of biochar hindered Pb transfer from roots to shoots and reduced Pb enrichment in plant, similar with previous findings by Zulqurnain Haider, et al. 63 and Medyńska-Juraszek, et al. 64. It is possible to restrict the migration of heavy metals to plants by adding a suitable amount of biochar to the soil. This is because heavy metals in the soil may be changed from reactive to more inert forms by using biochar, leading to a decrease in the transport and bioaccumulation of these metals 65,66.
Through random forest analysis, various important predictors of root and shoot biomass in red clover treated with different doses of biochar were evaluated. The results showed that stem biomass was closely related to plant height, chlorophyll a, chlorophyll b and carotenoid contents, while root biomass was significantly related to root length, plant height, SOD activity and chlorophyll a content. Therefore, the ability of biochar to promote biomass accumulation may be an indirect result of increased photosynthetic pigment content. Studies have shown that exposure to Pb can harm the intricate structures present within chloroplasts, causing the breakdown of thylakoids, which hampers gas exchange and the production of photosynthetic pigments. 67,68. However, the addition of biochar can alleviate this phenomenon and improve the photosynthetic activity of plants, thus promoting plant growth and biomass 69. On the other hand, shoot biomass was significantly correlated with Pb-related parameters, such as shoot and soil Pb contents, while root biomass was significantly correlated with soil Pb contents, root and shoot and transport factors. These results can be connected to the research of Ma, et al. 44 that biochar can adsorb Pb from soil, thereby reducing lead transport and accumulation in plants to mitigate Pb toxicity.