At lower concentrations, Cd, NP, Cd and NP treatments had no significant toxic effects on plant growth, even had some promoting effects. At higher concentrations, Cd, NP treatments had significant effects on plant growth. This is a typical hormesis phenomenon (Calabrese, 2005). When the concentration of Cd was 5 mg/L and the concentration of Cd + NP was 5 + 0.1/1 mg/L, the plant died in the course of cultivation due to severe poisoning. The results suggested that high Cd and NP stress inhibited plant growth, which agrees with previous studies (Wang et al., 2018). We observed plant poisoning symptoms in the experimental process: leaves from the edge to the center gradually yellowing, and then necrosis, plant root growth inhibited or even rot necrosis. The higher the concentration of the poison applied, the sooner the plant will develop these toxic symptoms. This is similar to a study by (Liu et al., 2019a). An important cause of Cd poisoning is its high chemical similarity with functional active ions (especially Zn, but also Ca and Fe) at active sites of enzymes and signal components, leading to their displacement from proteins (Gallego et al., 2012). Thus affect the absorption of many main nutrients and micronutrients, inhibit plant growth. As a hydrophobic organic compound, NP easily penetrates the cell membrane and acts on the cell's internal structure, thus stunts cell growth (Bhandari et al., 2021). A study suggests that (Shi et al., 2017), Cd and NP can affect the absorption of organisms to each other. Low concentration of NP can offer carbon source for Cd removal. Low concentration of Cd can change the hydrophobicity of cell membrane and increase the absorption of organic matter. However, high concentration of Cd-NP combined pollution can damage the permeability of cell membrane and affect the growth and metabolism of organisms.
Chlorophyll is the material basis of photosynthesis, and its content can be used as a physiological index to measure the stress resistance of plants, and is an important indicator of plants under environmental stress (Du et al., 2018). Stress of heavy metals inhibited the photosynthesis of plants. In this study, the chlorophyll a, chlorophyll b and carotenoid contents in leaves of Cd treated groups were lower than those of control group. However, both NP alone and NP + Cd combined treatments showed a tendency of low concentration promoting chlorophyll synthesis and high concentration inhibiting chlorophyll synthesis, and the decrease was deeper than that of Cd or NP alone. It's possible that the two poisons have a synergistic effect on the plant. The results of this study are different from those of others, Shi et al. (Shi et al., 2018) studied the effects of combined Cd and NP stress on the growth and physiological indexes of ryegrass. Results showed that 0–5 mg/L NP had no significant effects to the chlorophyll contents, but 0–10 mg/L Cd had a significant effect to the chlorophyll contents. The change trend of the chlorophyll contents of ryegrass under joint stress was the same as that under Cd-only stress. Therefore, the difference of joint toxicity effect is related to the nature and concentrations of toxicants and the treatment time, and the tolerance of different plant species and different growth stages to toxicants is also different.
Under stress or adverse environment, plants will produce reactive oxygen species (ROS) such as superoxide free radical (O2 −), hydroxyl free radical (OH −) and H2O2. ROS can cause oxidative damage of lipid, protein and DNA in cells and induce oxidative stress. Therefore, the production and removal of H2O2 in plant tissues under adversity can indirectly reflect the degree of plant damage caused by toxicants and the resistance of plants. When a large number of free radicals accumulated on the membrane, MDA as a reaction product began to accumulate. MDA can reflect the degree of cell damage, cell metabolism disorders, resulting in decreased plant biomass and leaves chlorosis and other toxic symptoms (Malik et al., 2021). H2O2 is an active oxygen molecule in organism and an important signaling molecule in response to stress, which is involved in plant resistance response (Yang et al., 2018). Our study showed that under Cd and NP alone or combined treatment, the MDA content at low concentration (0.01 + 0.1 mg/L) was not significantly different from that in control group, but significantly decreased under high concentrations stress. Under compound stress, the MDA content was lower than that of single stress, it could be the combination of pollution making it more toxic. This indicated that H. dubia had some resistance and adaptability to Cd and NP at low concentrations. Cell protection systems were activated at high concentrations, which eliminated the free radicals and reduced the MDA content. Under Cd-NP stress, the H2O2 content in the treatment groups was higher than that of the control. The increase of H2O2 content indicates that the original balance of active oxygen metabolism is destroyed.
In bad environment, plants have formed a set of effective oxidation defense mechanisms and highly complex detoxification mechanisms, namely enzymatic and nonenzymatic antioxidant protection systems (Wei et al., 2009). They protect plants from ROS. CAT, POD and SOD are three important defense enzymes in plants antioxidant systems (Shakir et al., 2018). SOD as the first line of defense for plant cells to eliminate ROS and catalyze the disproportionation of O2− to form H2O2 and O2. CAT is a heme-containing protease that catalyzes H2O2 produced during photorespiration, mitochondrial electron transport and fatty acid oxidation, and converts H2O2 to H2O and O2. POD is an enzyme that uses H2O2 as an electron acceptor to catalyze the oxidation of substrates to produce H2O (Liu et al., 2019b). In this study, 14 days after Cd and NP treatment, the POD activity in the leaves of H. dubia was significantly increased at high concentrations (≥ 0.5 mg/L), especially in the joint treatment. The CAT activity in 0.5 + 0.1 mg/L and 0.5 + 1 mg/L was significantly lower than that in the single exposure groups. The activity of SOD decreased at first and then increased in the alone treatment, and decreased significantly in the joint treatment. In general, alone and joint exposure of Cd and NP resulted in the decrease of SOD and CAT activity and the increase of POD activity in leaf tissues of H. dubia. It indicated that the antioxidant systems were damaged to some extent, and the combined stress aggravated the plant injury, caused the plant cell metabolism disorder. Previous studies have also demonstrated that Cd and NP combined stress can significantly increase oxidative stress levels in Chlorella sorokiniana, and the mixed toxicity showed synergistic effects of SOD and CAT activities (Wang et al., 2018). POD plays a major protective role, which can make up for the decline of CAT activity when plants are subjected to oxidative stress, and plays a major role in scavenging reactive oxygen species, the change trend of H2O2 content in plant leaves was the same as that of POD activity. SOD activity was negatively correlated with Cd content in the leaves of H. dubia, which was similar to the results of Yang (Yang et al., 2017). The decrease of CAT activity under single stress and the positive response of CAT in low concentration combined exposure may be related to the synergistic effect of Cd and NP on H. dubia. Because of the strong oxidative stress, the plant was damaged, the protective systems were damaged and the protein was inactivated in the higher concentration of the combined poison. The results also showed that CAT played different roles under different pollutant stress, and enhanced CAT activity could help to eliminate excessive H2O2 in a certain concentration range. But when the concentration exceeded this threshold, the activity of CAT was inhibited when the oxidative stress caused by high concentrations of poison exceeded the resistance of enzyme. The increase of POD activity indicated that Cd + NP stress could accelerate the senescence of H. dubia. Our results showed that POD is more sensitive than CAT to remove H2O2 produced by Cd and NP, and H2O2 is eliminated mainly by POD or other enzymes (such as GSH) and nonenzymes (such as ASA).
Soluble protein in cells are mostly enzymes involved in metabolism and are an important indicator of metabolic intensity in plants (Wang et al., 2013). In this study, 14 days after being exposed to NP alone and Cd + NP, the soluble protein content in plant leaves increased first and then decreased with the increase of toxic concentrations. The soluble protein content was lower at the combined stress than that of the single treatment. This is similar to the findings of Mao and Yang (Mao and Yang, 2019). The decrease of soluble protein content in plant tissues indicated that the stress degree was enhanced under combined stress.