Responses of electrical conductivity and MDA content to elevated CO2 and/or Pb stress
Electrical conductivity and MDA content are important indexes to measure the degree of membrane permeability and membrane lipid peroxidation. As one of the important products of membrane lipid peroxidation, MDA can aggravate membrane damage. In our study, EC decreased electrical conductivity compared with control; Electrical conductivity and MDA content increased under Pb stress, thus Pb stress is toxic to cells, leading to cell membrane system damage, loss of selective permeability, membrane dysfunction, intracellular electrolyte extravasation; Under compound treatments, compared with AC, electrical conductivity and MDA content were nearly not changed, but MDA content increased under EC + H. EC + L and EC + H decreased electrical conductivity and MDA content compared L and H, the results show that high concentration of CO2 could significantly alleviate oxidative damage caused by lead stress and maintain the stability of cell structure and membrane permeability.
Responses of SOD, POD, CAT activities to elevated CO2 and/or the Pb stress
SOD is a metal enzyme in the core of antioxidant enzyme system. POD is a metal enzyme containing Fe, which is involved in cell division, respiration, photosynthesis and auxin. CAT is the terminal of a series of antioxidant enzymes in the process of biological oxidation in plants, which can remove excessive H2O2 in cells, maintain the balance of ROS metabolism and protect the integrity of cell membranes. Only the coordination of SOD, POD and CAT can better resist injury.
In this study, SOD, POD and CAT activities in rice seedlings increased under EC, compared with AC. The studies have shown that plants growing under high concentrations of CO2 can regulate the photosynthetic electron conduction system and synthesize more NADPH (Lichtenthaler 1987), and the synthesized NADPH can be used for the ascorbate-glutathione cycle (Rao et al. 1995), which keeps ascorbic acid and glutathione in a high reduction state in plants, thus inducing the activities of antioxidant enzymes such as SOD (Foyer et al. 1994a, b); Under Pb stress, SOD, POD and CAT activities were almost increased compared with AC, except POD activity under H, it may be caused by the induction of a large number of reactive oxygen species under stress; Under composite treatments, compared with AC, SOD, POD and CAT activities were all increased. Compared with L and H, SOD, POD and CAT activities were also almost increased, except SOD activity under EC + L, which decreased compared with L. High concentrations of CO2 could enhance the resistance of rice to oxidative damage under Pb stress by increasing the higher activities of protective enzymes.
The response of metabolom to elevated CO2 and/or the Pb stress
To elevated CO2
Studies have shown that sugar, starch and amino acid contents of soybean increase under high concentration of CO2 (Gillespie et al. 2012). In our study, most of the contents of sugars and polyols increased, the increase of glucose glucose-6-P, glucose-1-P, fructose and fructose-6-P contents maybe caused by the efficient photosynthesis and glycolysis metabolism changes under elevated CO2. Mannitol, D-talose, xylitol, xylose, ribose, galactinol and raffinose also increased. Mannitol relates to fructose and mannose metabolism, and galactinol relates to galactose metabolism, these metabolic processes were all enhanced by EC; Related to amino acid metabolism, glycine, aspatic acid, serine, alanine, L-allothreonine and oxoproline contents were all increased, but glutamic acid content decreased. Glutamic acid is the precursor of chlorophyll a, and chlorophyll b evolved from chlorophyll (Ito et al. 1994; Woodward et al. 1990). The change of the glutamic acid content in plants under high CO2 may be related to photosynthesis (Gong et al. 2013). It is possible to synthesize more photosynthetic pigments by consuming glutamic acid to increase the photosynthetic intensity. Oxoproline relates to arginine and proline metabolism, which was also changes under EC; High CO2 concentration leads to a decrease in citric acid content while an increase in oxalic acid content (Miyagi et al. 2011). But we found, citric acid, succinic acid and L-malic acid (the downstream products of TCA cycle) contents in rice leaves increased, maybe related to the metabolic turnover of photosynthesis and dark respiration under EC. The increase of CO2 concentration can regulate the production of secondary metabolites in plants (Idso et al. 2001). In our study, dehydroshikimic acid, salicylic acid, ferulic acid, 4-hydroxycinnamic acid and benzoic acid related to secondary metabolism, the contents of them were all increased by EC. Oxalic acid and D-glyceric acid were also increased. Synthesis and transport of organic acids might be a special adaptation process to EC conditions. This assumption will need to be further investigated. In this study, linolenic acid and linoleic acid increased by EC, but stearic acid decreased, indicated that fatty acid β-oxidation was changed under elevated CO2 environment.
To Pb stress
The increase of soluble sugar contents is a typical stress response in plants under heavy metal stress. A series of stress resistance reactions occur in plants, such as the production of reactive oxygen species, signal transduction, and proteins and metabolites related to stress resistance, and the maintenance of these reactions requires a large amount of energy provided by carbohydrate compounds. Zeng et al. (2020) found, under cadmium stress, levels of most carbohydrates were down-regulated in the metabolomics study of two different varieties of indica and hybrid rice grains. But in this study, in rice leaves, sugar and polyol metabolism was enhanced, glucose, glucose-6-P, glucose-1-P, and fructose (related to glycolysis), mannitol, D-talose, xylitol, galactinol, raffinose, melibiose and trehalose increased under L and H, in addition, Fructose − 6-P and cellobiose also increased but only under H. Myo-inositol decreased under L, but increased under H. Ribose increased under L, but decreased under H. Xylose decrease under L and H. Sugar metabolism is associated with amino acid and organic acid metabolism, so it will indirectly affect the overall metabolic system of plants. Galactinol and melibiose relate to Galactose metabolism, trehalose and cellobiose relate to starch and sucrose metabolism, these metabolic pathways were all enhanced under Pb stress. Raffinose is an oligosaccharide with the function of storing energy and can be a protective agent under stress. The synthesis of raffinose involves the formation of myo-inositol, which is not only an important osmotic protective substance, but also is involved in a variety of metabolic regulation, such as the regulation of plant cell stress resistance, the promotion of seed dehydration and the regulation of plant auxin, and also participates in the synthesis of cell wall (Conde et al. 2015; Loewus and Murthy 1964; Madden et al. 1985). In this study, Myo-inositol and Raffinose were all increased under H as an important metabolic regulator.
Free amino acids are not only the basic unit of protein synthesis, but also have a significant effect on plant resistance to stress (Less and Galili, 2008). Amino acid metabolism is enhanced and the small molecule amino acids accumulate, contributing to improve osmotic adjustments and maintaining cell membrane stability (Widodo et al. 2009). It maintains osmotic pressure of plant cells and can be used as a mediator for heavy metals and form metal complexes (Bottini and Festa 1996). Heavy metal stress inhibits normal nitrogen metabolic pathways in plants. Free amino acids, as an important form of N assimilates in plants and the main transport form, can reflect the supply capacity of N assimilates (Zhao et al. 2019). In our study, amino acid metabolism was enhanced, glycine, aspatic acid, serine, alanine, valine, L-allothreonine and oxoproline contents were all increased. Alanine and valine are glucogenic amino acids closely related to pyruvate metabolism. They accumulated significantly under Pb stress, suggesting that they might promote glyconeogenesis as an effective transformation pathway to resist Pb stress. The stress response of alanine is mainly reflected in the regulation of intracellular environment pH, and the increase of alanine content may be caused by the reduction of protein synthesis rate and the slow reaction of alanine transaminase after heavy metal stress (Xu et al. 2012); oxoproline is related with proline and arginine metabolism, the accumulation of proline can decrease intracellular osmotic potential, balances protoplast osmotic pressure, protects enzyme activity, and reduces soluble protein precipitation (Yang et al. 2017). Arginine has the function of storing N and is also the precursor of polyamines and nitric oxide, which are involved in almost all physiological and biochemical processes, including growth, development and stress resistance (Crawford 2006; Modolo et al. 2006).
Some abiotic stresses can induce plants to secrete and accumulate organic acids, which can promote the activation and absorption of mineral elements in soil, and thus have the function of regulating plant nutrition (Wang et al. 2018; Yang et al. 2017), also they are vital osmotic adjustment solutes (Yang et al. 2017). In our study, citric acid, 3-phosphoglycerate and oxalic acid contents increased under Pb stress. As the intermediate products, 3-phosphoglycerate and citric acid relate to glycolysis and TCA cycle, the dehydrogenation process in such reaction is accompanied by NADH formation, which is essential to preserve the antioxidant capacity of cells (Petrat et al. 2003). Also, 3-phosphoglycerate relates to fructose and mannose metabolism, that all can provides energy for plants to resist stress. Organic acids contents are important metal ligands by participating in physiological metabolic processes such as absorption, transportation, storage and detoxification of heavy metals, the ionic metals are transformed into chelate states with low toxicity or non-toxicity, so as to reduce the toxic effects of excessive metals on plants (Ma et al. 2001). Oxalic acid has also been identified as a differential metabolite in several metabolomic studies under heavy metal stress (Lyubenova et al. 2013; Zeng et al. 2008), but only in root of plants. In our experiment, oxalic content of rice leaves also increased under L and H, speculation in oxalic acid is a major ligand of Pb, induced by Pb stress, thus reducing the Pb and the combination of the important enzymes in the cell, the opportunity to relieve the Pb in the leaves of poison. Related to secondary metabolism, salicylic acid, ferulic acid, 4-hydroxycinnamic acid and benzoic acid contents increased. The increase of this important secondary metabolites’ contents related to strong antioxidant capacity of plants. In our study, D-glyceric acid content only increased under H and dehydroshikimic acid content decreased under L and H.
β-oxidation is the primary manner of fatty acid decomposition, which provides a large amount of energy needed for life activities, thus playing an important role in plant stress responses (Cooper et al. 1969). In our study, among the fatty acids, linoleic acid, palmitic acid and stearic acid contents decreased under H. This showed that a severe Pb stress environment severely affected fatty acid metabolism, maybe the fatty acid β-oxidation was enhanced. This is related to the higher metabolic energy consumption of plants under Pb stress.
To Elevated CO2 and Pb stress
Under compound treatments, compared with AC, the overall metabolic trend was similar to that of EC treatment. But we can also see the difference, mainly reflected in severe Pb stress, such as, salicylic acid content decreased and benzoic acid showed no significant change under EC + H, cellobiose and palmitic acid contents also decreased. That means when rice seedlings were treated under EC and Pb stress together, high concentration of CO2 play a dominant role, especially under mild Pb stress (EC + L), but not severe Pb stress (EC + H). Ethanolamine is important for synthesis of choline, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in plants. These two phospholipids are the major phospholipids in eukaryotic membranes, so it is important in plants growth and developmental processes (Kwon et al. 2012). In our study, ethanolamine content also increased under EC + H, this may be related to its protective effect on cell membranes under adverse conditions.
Under EC + L, compared with L, high CO2 concentration promoted the rise of glucose-1-P, mannitol, D-talose, xylitol, ribose, xylose and galactinol contents, but decreased contents of glucose, fructose, melibiose, myo-inositol, cellobiose and trehalose. Perhaps, more glucose was converted to other sugars, so as to resist to lead stress at the cost of decreasing glycolysis pathway; Related to amino acid metabolism, EC + L increased aspatic acid and serine contents, but decreased glycine, valine, L-allothreonine, glutamic and oxoproline contents, perhaps means under high CO2, amino acids are no more important for osmotic regulation under mild lead stress. The decrease of oxoproline content perhaps relates to the metabolism of glutamic acid and proline; Contents of citric acid, succinic acid and L-malic acid were all increased, just like changes under only EC treatment, they are important intermediate products of the TCA cycle. Plants can prevent, reduce, or repair damage caused by unsuitable environments using a series of physiological and metabolic reactions. The enhancement of the TCA cycle in response to Pb stress could have been due to the increase in some intermediate metabolite levels, and its dehydrogenation process is accompanied by NADH formation, which is essential to preserve the antioxidant capacity of cells and favorable for N metabolism (Petrat et al. 2003). Also, Citric acid and malic acid are scavenged active oxygen species (Li et al. 2017). Under high concentration of CO2, accumulation of those organic acids indicated that it could adapt to Pb stress by maintaining a high antioxidant state, but the above process occurs only under L, not H treatments. D- glyceric acid content was also increased, but 3-phosphoglycerate decreased. D-glyceric acid can be converted from 3-phosphoglyceric acid and enter the Calvin cycle to promote photosynthesis under high CO2. It should be noticed that dehydroshikimic acid, salicylic acid and 4-hydroxycinnamic acid (relate to secondary metabolism) were all increased under EC + L. Those may be the important regulatory substances that play a major anti-stress function. Salicylic acid (SA) is significantly accumulated, that has an important role in plant Pb tolerance. it can induce or enhance the antioxidant system to remove excessive ROS in plants, reducing the degree of cell membrane lipid peroxidation, improving cell metabolic activities and alleviating the inhibition of plants under stress (Zhao and Zhang 2013). Our study confirmed that under high concentration of CO2, the accumulation of SA was also an important mechanism of Pb tolerance under only L, but not H; In addition, high concentration of CO2 promoted the reduction of palmitic acid contents under mild Pb stress, which may be involved in β-oxidation, so as to provide energy for rice to resist Pb stress, also, linolenic acid and linoleic acid increased, they are unsaturated fatty acids. Fatty acids are important components of the cytomembrane and Pb stress may damage its structure and decrease the fatty acid content, which allows the cell contents to spill out (Salama and Mansour 2015). Studies have demonstrated that plants maintain fluidity of the cell membrane by fatty acid desaturation (Gao et al. 2010). That may be an effective way of adapting to Pb stress.
Compared with H, EC + H increased most of sugars and polyols contents, include glucose, glucose-1-P, fructose, fructose-6-P, mannitol, D-talose, xylitol, xylose, ribose and galactinol except that myo-inositol and trehalose contents decreased. Also, most of amino acids (include glycine, aspatic, serine, valine and L-allothreonine) contents increased, but glutamic decreased. Maybe, under severe Pb stress, more osmotic regulatory substances (sugars, polyols and amino acids) were needed. Compared with AC, myo-inositol content of rice seedling leaves increased as a protect matter under H, but under compound treatments, myo-inositol decreased, compared with single Pb stress. That means under EC, myo-inositol may be consumed with the increase of sugars metabolism; Among organic acids, Dehydroshikimic acid, ferulic acid and 4-hydroxycinnamic acid contents increased, but salicylic acid content decreased. That means under EC + H, secondary metabolites still play an important role in anti-stress. Dehydroshikimic acid can promote the formation of shikimic acid, about 20% of the C fixed by green plants is metabolized by shikimic acid, resulting in aromatic amino acids, plant hormones, phenols and other substances. Some of these substances can act as signaling molecules to regulate plant development, and some give plants the ability to resist injury and oxidation (Herrmann 1995; Wilson et al. 1998). The results suggested that EC enhanced tolerance by regulating C metabolism, causing more C to flow to the shikimic acid pathway and producing more downstream substances necessary for normal plant growth. In addition, ethanolamine content was more increased under EC + H, compared with H. That means, under EC, ethanolamine maybe the important protective substance to resist severe Pb stress. Oxalic acid was also increased as a major ligand of Pb.