4.1 Lead effects on the growth of K. paniculate
Growth morphology and biomass changes are the ultimate external form of plant growth adapted to the environment. Heavy metal stress inhibits plant growth, resulting in smaller leaf area, dwarfism and reduced plant biomass. In general, plants have a toxic response at a low level of heavy metals, and the biotoxicity of heavy metals is 10–100 times higher in hydroponic tests than in soil tests (Jinwei et al. 2008). In this test, Pb concentrations below 400 mg/L accelerated the growth of K. paniculate compared to the CK. When Pb concentrations were above 600 mg/L, growth was significantly inhibited. However, the plants did not show mortality, but adopted a set of physiological and biochemical tolerance changes to tackle the harm from Pb.
The roots serve as the site of direct exposure to heavy metals. In this experiment, the number of fine roots decreased with increasing stress concentration, the average root diameter decreased, and the specific surface area decreased in response to the lead stress. However, under growth-promoting stresses of 200 and 400 mg/L, the number of fine roots increased, and the specific surface area did not change significantly. It could be that K. paniculate actively adapts to lead stress by increasing biomass and root share in adversity, and improving nutrient and water extraction capacity. The increase in mean root diameter may provide protection of the root cells from heavy metals toxicity by increasing the thickness of the epidermis and non-protoplast barrier (Cabot et al. 2014, Lux et al. 2010, Ryser &Emerson 2007). At the same time, root endothelium strengthening and the Kjeldahl band barrier are also essential mechanisms of root resistance to heavy metal ions (Vaculik et al. 2012). Usage of physical and physiological avoidance strategies to intercept most of the heavy metal contaminants in the roots showed that K. paniculate can adapt to Pb contamination to some extent.
The root zone in K. paniculate accumulated more than 80% of the lead. Studies have also shown that plants have high metal tolerance and can usually accumulate and fix large amounts of heavy metals in their roots, weakening the toxic effects on the plant body by preventing them from transferring from the subsurface to the ground (Cai-Ying et al. 2005). Overall, it seems that the transport of all parts of K. paniculate is relatively small, which is no more than 10%, and its above-ground parts have a weaker ability to accumulate Pb than hyperaccumulator plants, but with its huge biomass, the amount of enrichment should not be underestimated. Meanwhile, the K. paniculate roots stressed by the high Pb concentration (1200 mg/L) accumulated 3200 mg/kg of Pb, and no mortality occurred, which is sufficient to verify the high tolerance property of K. paniculate to Pb.
4.2 Defense mechanisms of K. paniculate leaves under lead stress
High concentrations of Pb stress inhibited plant leaf growth, and this decreasing trend may be related to the production of reactive oxygen species (ROS) (Bhaduri &Fulekar 2012). SOD, POD and CAT are essential components of the plant antioxidant system, scavenging excess O2− and H2O2 and their damage, inhibiting enzyme activity, poisoning membrane lipid peroxidation, and impairing the normal osmoregulatory capacity of cells (Bankaji et al. 2015). In this study, K. paniculate leaves did not show a single decline in enzyme activity. The CAT, POD activity of the leaves showed a trend of increasing and then decreasing, and they respectively reached the turning points of stress amounts of 400 and 600 mg/L. The results might attribute to the stimulation of antioxidant enzyme activity of K. paniculate within the threshold of lead stress, but as the stress concentration increased, the enzyme activity system of K. paniculate was disrupted, and the enzyme activity decreased. Similar to what previously had been reported (Zhengzheng et al. 2007), SOD activity maintained an upward trend to remove the harmful effects of reactive groups in the plant. In conclusion, K. paniculate reduces the toxicity of peroxides mainly by increasing leaf SOD activity and maintaining CAT, POD activity. The MDA content of K. paniculate leaves were at a stable levels under Pb 200–400 mg/L treatment, and significantly increased after the toxic effects of Pb stress became apparent. This might cause changes in the membrane structure of the leaf cells (Mahdavian et al. 2016, Vallee &Ulmer 1972). While Pb stress causes membrane lipid peroxidation damage in leaves, it also induces a response of antioxidant enzymes, antioxidants, and other resistance mechanisms in K. paniculate, leaving the total antioxidant capacity of K. paniculate leaves at a high level and enhances the overall antagonistic capacity of K. paniculate. This phenomenon, similar to the elevated soluble protein content, balances the osmotic potential between the cytoplasm and the vesicles, promotes the removal of reactive oxygen species in K. paniculate. It enables the normal proceeding of physiological activities such as cellular metabolism in K. paniculate, maintains the osmotic balance of cells and protects them from the toxicity of heavy metals (Peralta et al. 2001). It may be one of the main resistance mechanisms in K. paniculate.
Chloroplast chlorophyll content reflects the plants' photosynthetic capacity and directly affects their growth and development (Jain et al. 2009). Leaves in the treatment group above 1000 mg/L showed localized yellowing and black spots, similar to the response of many heavy metal-tolerant plants after suffering from excessive stress, including Robinia pseudoacacia(Yakun et al. 2016) and Koelreuterie paniculate (Zhang et al. 2019) ,etc. The toxicity of lead may affect the synthesis of prochlorophyll reductase and amino-γ-ketovaleric acid, hindering the chlorophyll synthesis process and leading to a decrease in chlorophyll content (Yu et al. 2010). However, high chlorophyll a/b ratios suggest that the greater the stacking of cystoid bodies, the lower the photoinhibition and the more efficient plants are in using sunlight energy (Feixiang et al. 2012). In this study, the a/b ratio content of stressed plants still fluctuated between 78.14% and 104.86% compared to the CK group. Despite causing some damage, K. paniculate was still able to maintain its growth by stabilizing chlorophyll a/b values to improve light energy use efficiency under high levels of lead stress (Yan Ao.lei et al. 2010).
4.3. Microcosmic structure response of K. paniculate under lead heavy metals stress
Cell wall fixation and vacuole-dominated distribution of soluble components are two main ways to detoxify heavy metals in plants(Hall 2002). The same conclusion was obtained in this experiment, and the distribution pattern of lead in the subcellular fraction of K. paniculate was seen in the subcellular fraction of K. paniculate tissues and in the relationship of cell wall > soluble fraction > organelle content fraction.
In all tissues of K. paniculate, the concentration of Pb2+ distributed in the cell wall fraction always occupied the largest proportion, and the increase of lead ions in the cell wall was even more obvious. The cell wall contains polysaccharides such as pectin, cellulose, hemicellulose, and protein, and many pro-metal ion coordination groups can complex with positive-valent metal ions in an inactive state. The cell wall plays an important role in the cumulative fixation of heavy metals and the reduction of their toxicity. After the concentration of lead ions increases and the active sites in the cell wall are saturated and occupied, soluble fractions such as vesicles will absorb metal ions and thus reduce the toxic effect of lead.
Employing transmission electron microscopy (TEM), the root cells of K. paniculate under lead stress were observed: cell walls in the plants of group CK were smooth and well-organized; with the increasing concentration of Pb, the distribution of substances in each cell tissue became well-defined, and apparent aggregation effects were observed in the soluble components of the cell wall and vesicles. In addition, the cell interstitial space also occupied a certain proportion of the content in the high concentration group F. The cell wall is the first barrier for extracellular substances to enter the cell. It contains a variety of polysaccharides and is rich in carboxyl, aldehyde, amino, and other metal-friendly coordination groups, which can be easily complexed with and immobilized heavy metals(Ghori et al. 2019, Pan et al. 2019). When the heavy metal ions bound to the cell wall reach saturation, the thick wall of Pb ions interwoven by the cell wall dextran can block most harmful heavy metals outside the cell and stay in the interstitial cell space. Depending on the loading level, excess heavy metal ions loaded on the cell wall leach into the cell and are transferred to the vesicles, where they are complex with organic acids and inorganic salts(Cosio et al. 2004).
Fourier transform infrared spectroscopy (FTIR) is a technique for structural analysis based on the vibrations of functional groups and polar bonds in compounds(Bosch et al. 2006). In this experiment, 2920 cm− 1 of -CH, -COO and 1630 cm− 1 of C = O may be more associated with the breakage of polysaccharide-rich peptide chains in the cell wall, combined with the widespread carboxylic acid and ketone groups in their secreted polysaccharides to reduce the toxicity of lead to K. paniculate (Xue et al. 2019). Compared to CK, especially for treatment B, the root promotion effect may attribute to the increased secretion of amino acids and carboxylic acids in the cell wall and vesicles of K. paniculate, which increased the binding of polyester polysaccharides and heavy metals, and incidentally promoted the plant growth. This is consistent with the promotion of plant growth at low concentrations (Clemens 2001).
When stress levels increased, the peak absorbance of the above wavelength showed a downward trend. It could be that the hydroxyl group of rhizome and stem cell wall was complexed with Pb and the saturation of hydrogen bond decreases. At the same time, high Pb stress inhibited the root secretion of K. paniculate, organic acids, amino acids, polypeptide substances, and protein and root transport channels were affected. The absorbance of rhizome relative to leaf was significantly reduced in each peak spectrum change trend of 1030 cm− 1 value in the infrared spectrum of leaf tissue with the increase of Pb treatment concentration, and finally tended to stabilize. As the Pb concentration increased, the level of cell membrane peroxidation was deepened, and the peroxide products of aliphatic ketones accumulated in the leaves to enhance the resistance of plates to Pb, which caused the increase in 1065 cm− 1 values. The comparative analysis of the variation trend is 1030 cm− 1. When roots, stems, and leaves are under high concentration stress, leaf tissue can still show specific Pb adaptability. It is speculated that some of the toxicity matter might be left in the root in exchange for reducing the toxicity in other parts to ensure the relative balance of the whole plant's physiological metabolism, which is consistent with the physiological indexes of a plant root system.