Growth and production
Soil contamination with CO causes abiotic stress in plants (Tariq et al. 2020). In plants, stress can be physiological and biochemical, and it can have positive and negative manifestations. The results of the present study indicate a direct relationship between the amount of CO and the amounts of YP and RDM, but the relationship is inverse with respect to ADM (Table 1). These results are different from those reported by Plice (1948) and Adieze et al. (2012) in Trifolium sp, Centrosema sp and Panicum maximum plants, whose growth increased with low doses of CO of 1%, but decreased with higher doses. Other authors differ, stating that CO does not inhibit plant growth. On the contrary, they mention that low and high doses (7.9 to 238 g/kg CO) promote the production of RDM and YP in Lh grass (Alejandro-Córdova et al. 2017; Orocio-Carrillo et al. 2019), which is evidenced by a J-shaped hormetic stress response for both parameters. This positive response to stress in Lh can be explained by a natural evolutionary adaptation to stress conditions (Agathokleous et al. 2020). Rivera-Cruz et al. (2016) found that Lh can remain for up to three years in soils contaminated with 60 to 119 g/kg of TPH, self-regulating its growth even when used to feed cattle. It is known that grasses survive and tolerate CO hydrocarbons in the soil, due to their fasciculated root system with abundant fine roots attached to rhizomes emerging from stem stolons in contact with the soil (Merkl et al. 2005; Rivera-Cruz et al. 2016). Rhizomes extending over the surface horizon in tropical wetlands form an extensive root system together with plant tillers (Rodríguez-Rodríguez et al. 2016). The abundant root growth in Lh grass under stress conditions shows the existence of mechanisms that effectively regulate the concentration of hormones and H2O2 as chemical signals that regulate cell activities (Segura 2008; Quan et al. 2008). However, although grasses regulate CO-induced stress, cell death by physical contact of CO with the roots of Lh does not have a negative effect on the plant, in contrast with what has been reported in other plants in which the oil clogs root pores and disrupts water osmosis (Sangeetha and Thangadurai 2014) and the absorption of nutrients (Osuagwu et al. 2013; Tahseen et al. 2016). The resistance of Lh roots to oil-induced stress may be explained by the metabolic induction of antioxidant production and premature lignification, which thickens the root wall (Vanholme et al. 2012). Root thickening in the presence of CO was observed in Lh roots in this study (unpublished data).
The stimulatory effect of CO on the multiplication and growth of YP from the base of the stem and root system of Lh grass may be the result of the microbial degradation of hydrocarbons (Maier and Gentry 2015) and the consequent increase of nitrogen, phosphorus, potassium and sulfate in the soil (Rivera-Cruz et al. 2012; Trujillo-Narcía et al. 2018). The Gleysol soil used in this research was very fertile, containing high levels of total N (0.20%) and extremely rich in PO4-2 (82 mg/kg) and SO4-2 (39 mg/kg). According to Clarkson et al. (2000), these levels of available soil nutrients promote stomatal conductance and hydraulic conductivity in roots under stress conditions. There are claims that plants increase their growth in soil contaminated with CO because “petroleum auxins”, identified as naphthenic acids, improve crop yields, stimulate photosynthesis and increase protein nitrogen (Baker 1970).
H2O2 and antioxidants in Leersia hexandra leaf
The study of oxidative stress and the antioxidant response induced by hydrocarbons and inorganic elements of CO in plants is a complex affair. The effects of CO contamination on living beings have been studied mainly by considering how CO affects the soil. CO causes changes in temperature, hydrogen potential, electrical conductivity, water availability, and nutrient availability. It also favors the accumulation of heavy metals and toxic hydrocarbon metabolites derived from degradation and mineralization (Devatha et al. 2019; Odukoya et al. 2019). The results of the present study indicate a high concentration of H2O2L in YP in the presence of CO and a highly significant positive relationship between them (0.791**, Table 5). The increase in H2O2 is in agreement with the results of Burritt (2008), Teotia and Singh (2014), and Ahmad et al. (2020), but there is no agreement on the H2O2L*YP relationship. In the Krona variety of rye (Secale cereale) H2O2 increases with 12% of oil, but RDM and ADM decrease (Skrypnik et al. 2021). Ahmad et al. (2020) reported that 5 and 10% concentrations of hydrocarbons inhibit the production of leaf biomass in Brassica oleracea, but increases the concentration of H2O2 in leaf. Gutiérrez-Martínez et al. (2020) reported an increase of 16% in the concentration of H2O2 in the leaves of Phaseolus vulgaris and a decrease in the leaf biomass exposed to 1 µM of Cd.
The excessive accumulation of H2O2 in Lh leaf is thus a biomarker of oxidative stress. It is evidence that the plant metabolism regulates physiological processes of senescence by eliminating the production of H2O2 at the right place and time (Foyer and Noctor 2003; Bhattacharjee 2010; Hasanuzzaman et al. 2014) since high levels of ROS have a deleterious effect on plasma membranes (Esim and Atici 2015; Kaya et al. 2019). The positive relationship (0.812** and 0.678**, Table 5) between the concentrations of TPL and TFVL and the concentration of CO indicates that these substances can serve as stress biomarkers at high concentrations of CO in the soil. However, CATL increases only at low doses of CO (Table 2). TPL and TFVL contribute to stress resistance and interact with many enzymes, growth regulators, and antioxidants in the plant, although the latter is still under debate (Treutter 2010). These compounds are also mediators of ROS homeostasis in vacuoles, acting mainly as antioxidants (Agati and Tattini 2010). The results of the present study indicate an increase in TP in Lh leaf due to the effect of CO. Similar results were reported in Krona and Valdai rye with 12% of CO (Skrypnik et al. 2021) and in C. leucanthemum with 7.5% CO (Noori et al, 2012). TFVL also increase in the presence of CO, similar to the behavior of phenanthrene in Arabidopsis thaliana (Lukaszewicz et al. 2004; Fini et al. 2011) and Chrysanthemum leucanthemum with 7.5 to 10% of CO (Noori et al. 2012). The regulation of redox homeostasis in plants under polluted conditions is based on the activity of low molecular weight molecules of the enzymatic and non-enzymatic antioxidant systems (Skrypnik et al. 2021). Some authors assume that the enzymatic system provides is the metabolic process that provides the most effective protection against damage caused by ROS (Quan et al. 2008). In the present study, the positive correlation of TPL (0.575*) and TFVL (0.718**) with H2O2L (Table 5) confirmed that the enzymatic system may serve as a self-regulatory mechanism for the removal of H2O2 in YPL. However, Hernández et al. (2009) and Agati et al. (2012) indicate that the correlation between oxidative stress, ROS generation, and ROS removal activity by TP and TFV remains a controversial subject because the interaction between antioxidant enzymes, substrate (flavonoid), and H2O2 occurs only after the rupture of the tonoplast membrane. In the present study, the highly significant positive relationship between YP and TPL (0.845**) and TFVL (0.706**) (Table 5) suggests that these non-enzymatic antioxidants are stress signalers that contribute to YP growth. These compounds cause changes in the levels of 3-indoleacetic acid and the biosynthesis of LEA (late embryogenic abundant) proteins that bind to cell membranes and proteins of cell reproductive organelles (Matilla 2008).
In YPL, CAT was more sensitive than TP and TFV. The production of this enzyme was stimulated only at low doses of CO, showing a negative relationship with CO (-0.530*, Table 5). These results are similar to those reported by Al-Hawas et al. (2012) in Simmondsia chinensis. In that case, the concentration of CATL increased in plants exposed to 1% CO, but decreased when exposed to doses of 2 and 3%. In A. thaliana, CAT behaves in a similar way when the plant is exposed to phenanthrene; in Kandelia candle, when exposed to any of the 16 polycyclic aromatic hydrocarbons (Song et al. 2011; Liu et al. 2014); in Lolium perenne to phenol-polycyclic aromatic hydrocarbons (Malincka et al. 2021); and in Cynara cardunculus var altilis, when exposed to 100mM NaCl (Docimo et al. 2020). The inhibition of CAT in YPL exposed to CO indicates that the presence of oil disrupts cellular synthesis, which directly induces the accumulation of H2O2 in leaf, considering that this antioxidant is directly involved in converting H2O2 into water and oxygen under stressful conditions that cause ROS to accumulate (Glorieux and Calderón 2017).
H2O2 and antioxidants in Leersia hexandra root
Plant roots adapt to different soil conditions (López-Bucio et al. 2003). In the roots of Lh, the amount of synthesized H2O2 decreases according to the concentration of CO (-0.937**, Table 5). Similar behavior is observed in the root of Glycine max exposed to NaCl (Neves et al. 2010). An opposite behavior is observed in the roots of G. max exposed to Cd (Finger-Teixeira et al. 2010) and in Rumex dentatus, Euphorbia helioscopia, Cannabis sativa and Parthenium hysterophorus exposed to Pb and Cr (Ullah et al. 2019). These recent findings show there is variation in the synthesis of H2O2 in the root of different plants when exposed to stress. Each type of plant regulates the synthesis of H2O2 differently. H2O2 is a signal molecule involved in acclimation signaling that triggers stress tolerance (Gill and Tuteja 2010). The low concentration of H2O2 in the root of Lh exposed to CO suggests that it is a stress signaling molecule, but this concentration is sufficient for promoting plant growth and for preserving the permeability of the cellular channels that conduct calcium and potassium for cellular homeostasis (Foreman et al. 2003; Kwak 2003), cell cycle regulation (Mittler et al. 2004), growth and development (Foreman et al. 2003) and the timely translation of genetic signals (Quan et al. 2008).
The synthesis of root antioxidants as stress biomarkers behaved differently than YPL. In the roots, CO negatively affected TPR (-0.876**) and positively PALR (0.864**). The behavior of TPR whenLh is subjected to oil-induced stress is similar to that of Lens culinaris cv. Tub exposed to 0.5 mM Cu2+ (Janas et al. 2009). The opposite behavior is observed in C. leucanthemum exposed to 10% of CO (Noori et al. 2012). The significant negative relationship TPR*RDM (-0.897**, Table 5) suggests that TP possibly promotes increased root growth by contributing to control the endogenous levels of 3-indoleacetic acid that the plant requires for its growth (Engels et al. 2012). In the present study, Lh regulates the physiological balance of the roots under stress based on a significant and positive relationship TPR*H2O2 (0.817**, Table 5). The CAT in the root shows no relationship with H2O2R and RDM, but increased in roots exposed to 30, 60 and 90 g/kg of oil, compared to the control. This behavior is different than that observed in the roots of Lactuca sativa, Cichorium endivia, Apium graveolens, Petroselinum crispum, and Solanum melongena, but similar to the behavior observed in Lycopersicon esculentum exposed to 3-amino-1,2,4-triazole (Chioti and Zervoudakis 2017). Since CAT has been reported to be synthesized in the mitochondria (Willekens et al. 1995) and H2O2 is generated during mitochondria respiration (Giorgio et al. 2007), the physiological role of this isoenzyme in the roots of Lh could be to promote the reduction of H2O2 by oxidizing it and obtaining water and oxygen, as indicated by Glorieux and Calderon (2017). An outstanding result of the present study is the increase in the concentration of PAL in the roots of Lh. A highly significant positive relationship of this enzyme with CO (0.864**) and RDM (0.709**, Table 5) indicates the protective role it plays in the roots and as a stress biomarker. Similar behavior has been observed in the roots of G. max exposed to Cd (Finger-Teixeira et al. 2010). An opposite behavior has been observed in G. max and Z. mays exposed to dopamine (Guidotti et al. 2013; Siqueira-Soares et al. 2013). In G. max and Beta vulgaris treated with mannan oligosaccharide for the control of Meloidogyne javanica nematodes, where PAL is an indicator of resistance to stress (Débia et al. 2021). PAL biosynthesis has been reported to increase in Pea seedlings when ethylene was applied to the plants (Hyodo and Yang 1971), which suggests that ethylene is present in soil derived from the degradation of linear petroleum hydrocarbons (Fessenden and Fessenden 1983), which in contact with the roots of Lh induce root growth. PAL synthesis is important for plant defense and growth due to its role in the synthesis of the secondary metabolite phenylpropanoid, which plays a crucial role in the biosynthesis of monolignols, salicylic acid, phytoalexin, and flavonoids (Deng and Lu 2017; Odukoya et al. 2019).
Bacteria and removal of petroleum hydrocarbons
Various studies have identified that the rhizosphere of grasses used to remove TPH in soils contaminated with CO host an intense microbial activity, including bacteria that regulate plant growth (Arias-Trinidad et al. 2017; Rodríguez-Rodríguez et al. 2016). The increase in the population of Azotobacter spp (0.850**, Tables 2 and 5) in the rhizosphere of Lh growing in soil contaminated with CO is similar to the results reported in other studies of Lhexposed to 60-180 g/kg of TPH (Arias-Trinidad et al. 2017) and Paspalum virgatum exposed to 25 g/kg of TPH (Rivera-Cruz 2011). The increase in the population of Azotobacter spp in the rhizosphere of Lh in the presence of CO is a consequence of the ability of these bacteria to adapt to and resist stress conditions by forming cysts in response to changes in organic substances such as ethanol, n-butan-1-ol or β hydroxybutyrate (Aasfar et al. 2021). The adaptative capacity of Azotobacter spp explains its positive relationship with TPH removal (0.597*, Table 5). In contrast, Azospirillum spp and Pseudomonas spp showed no relationship with TPH removal, and their populations were higher in soil with NA. Under CO-induced stress conditions, Azotobacter may acquire plasmid-encoded catabolic genes through horizontal gene transfer processes (Maier and Gentry 2015). Sun et al. (2012) and Penton et al. (2013) mention that stress-adapted bacteria promote the interaction of proteins and DNA with polycyclic aromatic hydrocarbons such as naphthalene, fluorene and pyrene, which oxidize them and form metabolites that are incorporated into the catabolic pathways of the cell, releasing energy. The Lh rhizosphere induces the removal of 22 to 37% of TPH in soil containing between 30 and 90 g/kg of CO. The removal of TPH induced by PH has a positive relationship with RDM (0.601**) and the population of Azotobacter spp (0.592*). However, NA achieved a higher removal percentage of between 30 and 57.6%. The removal of TPH by PH from the grasses has been reported by various authors (Liao et al. 2015; González-Moscoso et al. 2019), who recognize that it is a long-term technology with the advantage of providing environmental services to living organisms. Other experimental results (González-Moscoso et al. 2019; Orocio-Carrillo et al. 2019) show thatLh can remove up to 76.6% TPH within six to eight months, but NA yields better results. These authors explain that the higher percentage of removal in non-rhizosphere soil may be because in the soil, the immediate source of organic carbon is petroleum hydrocarbons, while in the rhizosphere the microorganisms have two sources of carbon: root exudates (amino acids, auxins, ethylene, flavonoids, organic acids, palmitic acid, quercetin, strigolactone, and sugars exuded by the root) (Kuzmicheva et al. 2017), and petroleum hydrocarbons, which leads to a process of cometabolism in which microorganisms use both carbon sources. Furthermore, the colonization of the grass rhizosphere by Azotobacter and the inhibition of Azospirillum and Pseudomonas affect the production of root exudates, which can vary depending on the exposure to stress and other factors (Philippot et al. 2013; Pérez-Jaramillo et al. 2015). One of these factors is the amount of detached root cells, which are labile and easily degraded, that incorporate into the soil (Neumann and Römheld 2012). The roots of Lh have a high content of TFV and PAL (Table 2) and, according to Badri et al. (2013), these natural phytochemicals in the rhizosphere stimulate and inhibit a wide variety of microbial communities. In this regard, this study shows a significant positive relationship between AZT and TPR (0.868**) and with PALR (0.622**, Table 5).