Effect of C14 alkane on biomass of ryegrass
Biomass yield were measured to examine the effects of C14 alkane on the health of ryegrass. As shown in Fig. 1, the biomass yield of shoot and root of ryegrass gradually decreased with the increasing of C14 alkane concentration. The significantly decrease of shoot biomass was occurred at 0.2% C14 alkane treatment, while at 0.5% C14 alkane treatment a more dramatically inhibitory effect was observed and the percentage of inhibition was approximately 58.67% of control treatment. The ryegrass root biomass also significantly decreased when C14 alkane were added in soil, the percentage of decrease at 2% C14 alkane treatment was approximately 46.34% of control treatment. Toxic effects of organic compounds on plant growth and biomass have already been observed in many plants (Xie et al., 2017; Gao et al., 2018; Xi et al., 2018), which was consistent with this research.
Effect of C14 alkane on Cd concentration and accumulation of ryegrass
After different concentration of C14 alkane treatments, the Cd concentration and uptake amount in the shoot and root of ryegrass were shown in Fig. 1. The results indicated that the presence of C14 alkane in soil significantly affected the Cd concentration and uptake amount of ryegrass. Compared with control treatment, Cd concentration in shoot and root of ryegrass significantly increased at 0.1% C14 alkane treatment, then gradually decreased with the increase of C14 alkane concentration. When C14 alkane concentration increased to 1%, Cd content in shoot of ryegrass was significantly lower than that in control treatment. However, Cd content in root of ryegrass at 2% C14 alkane treatment presented no significant difference with control treatment.
With the increase of C14 alkane concentration, the variation trend of Cd uptake amount in shoot and root of ryegrass were similar with that of Cd concentration. The maximum value of Cd uptake amount was observed at 0.1% C14 alkane treatment, which was 30.17 µg·pot− 1 in shoot and 4.44 µg·pot− 1 in root. Compared with the control, Cd uptake amount at 0.1% C14 alkane treatment were increased by 69.9% in shoot and 48.8% in root. A similar phenomenon was found by Lu et al. that the pyrene addition significantly increased Cu, Cd, and Pb concentrations of both roots and shoots (Lu et al., 2014). However, Cd uptake amount gradually decreased with the increase of C14 alkane concentration. When the C14 alkane concentration increased to 0.5%, Cd uptake amount in shoots of ryegrass was significantly lower than that at control treatment. Compared with the control treatment, Cd uptake amount at 2% C14 alkane treatment were reduced by 85.1% in shoot and 47.8% in root. At 0.5% C14 alkane treatment, the decrease of ryegrass biomass was the main reason for the decrease of Cd uptake amount in shoot. When C14 alkane concentration increased to 1%, both biomass and Cd concentration had a significant effect on the decrease of Cd uptake amount in shoot. Gao and Zhu (2004) also reported that the concentrations of phenanthrene and pyrene above certain levels (133 and 172 mg·kg− 1 DW) decreased dry weight of 12 plant species.
Effect of C14 alkane on chlorophyll content of ryegrass
Chlorophyll a, chlorophyll b, and carotenoid concentrations in leaves of ryegrass grown in different concentration of C14 alkane contaminated soil were presented in Fig. 2. The results revealed that chlorophyll contents in ryegrass were gradually diminished with increasing concentration of C14 alkane, although differences in chlorophyll contents for some treatments were not significant. Compared with the control treatment, chlorophyll a, chlorophyll b, and carotenoid concentrations of ryegrass at 2% C14 alkane treatment were reduced by 16.37%, 23.16%, and 16.60%, respectively. The concentration of C14 alkane had a more significant effect on chlorophyll b than that on chlorophyll a and carotenoid.
Effect of C14 alkane on membrane lipid peroxidation and antioxidant enzyme activities of ryegrass
The MDA concentration was used as the general indicator of the extent of membrane lipid peroxidation (Ahammed et al., 2012). Elevation of the MDA contents indicated higher lipid peroxidation and over-production of reactive oxygen species (ROS) resulting from environment stress (Wang et al., 2006; Choudhary et al., 2011; Xi et al., 2018). The MDA contents in leaves of ryegrass grown in different concentration of C14 alkane contaminated soil were presented in Fig. 3a. Compared with the control treatment, the MDA contents in ryegrass significantly increased in all the C14 alkane treatments. When the C14 alkane concentration was less than 0.5%, the increase percentage of MDA concentrations were approximately 60% of control treatment, and no significant disparities among different C14 alkane treatments were shown. When the C14 alkane concentration increased to 1%, the MDA concentrations sharply increased. The highest MDA content was observed at 2% C14 alkane treatment, which was 160.2% higher than that in control treatment.
To prevent cell damage, antioxidant enzyme systems were elevated among plants for coping with environmental stress. SOD, CAT, and POD were three vital enzymes that can scavenge ROS in plant cells. SOD was the first defense against ROS since it can catalyze the conversion of superoxide radicals into hydrogen peroxide (Gao et al., 2018). The SOD activity in the leaves of ryegrass were shown in Fig. 3b. The SOD activity first gradually increased with an increase of C14 alkane concentration. The maximum value of SOD activity was observed at 1% C14 alkane treatment, which was increased by 4082% compared with that in the control. While, the superoxide radicals as well as other radicals can inactivate antioxidant enzymes (Gao et al., 2017). The SOD activity significantly decreased when the C14 alkane concentration were higher than 1%. It suggested that the superoxide radical production exceeded the ability of SOD to scavenge it.
The CAT activity was the primary H2O2 scavenging enzyme in plant cells, H2O2 can be reduced to H2O and O2 by it (Li and Yi, 2012). Compared with control treatment, the CAT activity had a slight increase when C14 alkane concentration was in the range of 0.1–1%, then significantly increased at 2% C14 alkane treatment (Fig. 3c). The CAT activity at 2% C14 alkane treatment increased by 121.8% compared with that in the control. The CAT results were consistent with the increase of MDA contents at 2% C14 alkane treatment as shown above. The elevated of CAT activity was associated with the increase of ROS, which was a signal molecule inducing the expression of CAT gene (Wang et al., 2012b).
POD was a defense enzyme that can scavenge highly toxic ROS major produced by SOD to plant cells (Zhou et al., 2016). POD had been reported to reduce H2O2 using phenolic compounds or flavonoids as donors of electron (Mitsou et al., 2006). The POD activity in the leaves of ryegrass at different C14 alkane concentration treatment was shown in Fig. 3d. Compared with the control treatment, the POD activity showed a slight increase trend at 0.1% C14 alkane treatment, then gradually decreased at the following two C14 alkane treatment (0.2% and 0.5%), and finally showed an increase trend again when the C14 alkane concentration was higher than 0.5% (Fig. 3d). The minimum value of POD activity observed at 0.5% C14 alkane treatment, which was 26.06% lower than that in control treatment. The results indicated that C14 alkane concentration just had a little impact on the POD activity of ryegrass. Base above analysis, we can conclude that SOD and CAT were the main antioxidant enzyme in ryegrass to prevent the C14 alkane stress.
Effect of C14 alkane on mineral nutrient elements uptake of ryegrass
The concentrations of mineral nutrient elements (K, Na, Ca, and Mg, Fe, Mn, Cu, and Zn) in shoots of ryegrass grown in different concentration of C14 alkane contaminated soil were shown in Fig. 4. The results indicated that the presence of C14 alkane in soil affected the uptake of all the measured elements. As shown in Fig. 4a, the K concentration in shoots of ryegrass showed a fluctuant trend with the increase of C14 alkane concentration. While the Na concentration decreased in the ryegrass grown in all the C14 alkane contaminated soil. The Ca concentration showed a slight decrease and no conspicuous changes was observed between different concentration of C14 alkane treatment. The Mg concentration obviously decreased in different C14 alkane treatments and showed a fluctuant trend with the increase of C14 alkane concentration. The Fe concentration significantly decreased at all the C14 alkane treatments compared with control treatment. The Mn concentration presented a gradually increase trend with the increase of C14 alkane concentration. The variation trend of Cu and Zn concentration in ryegrass were similar and showed no pronounced changes with the increase of C14 alkane concentration.
The correlation of chlorophyll content, antioxidant enzyme activities and mineral nutrient elements in the shoots was analyzed and listed in table S3. In addition, PCA was used to assess the relationship among the chlorophyll content, antioxidant enzyme activities and mineral nutrient elements (Fig. 5). The cumulative contribution ratio of PCA from the first two principal component were 74.5%. As the results showed, chlorophyll content and antioxidant enzyme activities were correlated with many mineral nutrient elements. Specifically, chlorophyll contents were positively correlated with K, Na, Ca, Mg, Fe and negatively correlated with Mn. MAD was positively correlated with Mn and negatively correlated with K, Na, Mg, Fe. POD activity was positively correlated with Cu and Zn. CAT activity was positively correlated with Mn, Zn and negatively correlated with K, Na. SOD was negatively correlated with K, Ca, Mg.
With the increased of C14 alkane concentration, the Mn concentration in the shoots of ryegrass gradually increased (Fig. 4f). Correlation analysis showed that the Mn concentration was significantly and positively correlated with MDA (Table S3 and Fig. 5). These results indicated that the oxidative stress of C14 alkane induced the increase of Mn concentration in shoot of ryegrass. Carvalho et al. (2019) also reported a similar phenomenon that Mn concentration significantly increased after Cd exposure. Previous studies had proved that supplemental Mn can reduce plants oxidative damage induced by Cd or salt (Cramer and Nowak, 1992; Rahman et al., 2016). Manganese played a vital role as cofactor in Mn-SOD and Mn-CAT, which participate in the plant’s defense against oxidative stress (Pittman, 2005). Meanwhile Mn can act as a scavenger of superoxide and hydrogen peroxide (Pittman, 2005; Millaleo et al., 2010). In the present study, Mn concentration was significantly correlated (p < 0.01) with CAT activity (Table S3, Fig. 5), which was consistent with previous researches (Rahman et al., 2016). However, no significantly correlation was observed between Mn concentration and SOD activity in shoots of ryegrass. In the 2% C14 alkane treatments, SOD activity in ryegrass dramatically decreased (Fig. 3b), which may be inactivated by superoxide radicals and other radicals (Ahammed et al., 2012). With the exception of 2% treatment, Mn concentration was significantly and positively correlated with SOD activity (r = 0.752, p < 0.01). We can conclude that the increased Mn concentration in shoots of ryegrass was a self-protection mechanism to reduce C14 alkane toxicity.
When the ryegrass was grown in the C14 alkane contaminated soil, Mg and Fe concentration in the shoot significantly decreased (Fig. 4de). The increasing uptake of Mn may be a reason for inhibiting the uptake of Mg and Fe (Heenan and Campbell, 1981). The results were similar to the previous studies which found that Mn concentration in the stem tissues of Lactuca spp. significantly increased but other essential micronutrients decreased under the stress of Cd (Ramos et al., 2002). Mg was an essential macro-element participating in the enzymatic reactions and photosynthesis as a structural component of the chlorophyll molecule (McSwain et al., 1976; Wang et al., 2017). Fe, which located mainly in the photosynthetic membranes, had a significantly influence on the structure and function of chloroplasts (Zembala et al., 2010; Liu et al., 2017). Combined with the results of the correlation analysis, we can conjecture that the decrease of Mg and Fe concentrations in shoots of ryegrass under C14 alkane stress was an important reason for the decrease of chlorophyll content and ryegrass growth.
Cd uptake amount in the shoot of plants was an important parameter for determining the efficiency of phytoremediation, since this part can be harvested easily and treated. Biomass and Cd content in plants were two important indices that affected Cd uptake amount. As indicated above, Cd uptake amount obviously increased at 0.1% C14 alkane treatment. The bioremediation of C14 alkane may decrease soil pH with a consequent solubilization of metals (Chen et al., 2004). In addition, Alkio et al. reported that PAHs can passively penetrate the root cell membranes of plants without any carrier, which can therefore facilitate the penetration of metal or metal complexes into the cells (Alkio et al., 2006). The C14 alkane might also facilitate the penetration of metal into the cells in a similar way as PAHs did.
However, Cd uptake amount gradually decreased with the increase of C14 alkane concentration. In order to reveal the primary factors that affected the Cd uptake, the relationship between the Cd uptake indices (biomass, Cd content, Cd uptake amount) and chlorophyll content, antioxidant enzyme activities, mineral nutrient elements were studied using an RDA (Fig. 6) and Pearson's correlation (Table S4). According to the RDA of the Cd uptake with respect to the mineral nutrient elements (Fig. 6a), the first two ordination axes explained 95.48% of the total variation in the Cd accumulation, with the first axis explaining 93.87% and the second axis explaining 1.61%. Cd uptake amount showed a positive correlation with Fe and Mg, but a negative correlation with Mn. According to the RDA of the Cd uptake with respect to the chlorophyll content and antioxidant enzyme activities (Fig. 6b), the first two ordination axes explained 88.25% of the total variation in the Cd uptake, with the first axis explaining 86.92% and the second axis explaining 1.33%. Cd uptake amount showed a positive correlation with chlorophyll content, but a negative correlation with MAD.
The growth characteristics of plants were one of the most important indicators used for the phytoremediation of contaminated soil (Zeng et al., 2020). As table S4 and Fig. 6 shown, significant positive relationships were discovered between biomass and chlorophyll contents. The chlorophyll contents reflect the photosynthesis ability of plants, which was one of the most essential processes of plant growth and development (Ahammed et al., 2012; Zeng et al., 2020). Meanwhile, biomass was positively correlated with K, Na, Ca, Fe, Mg. As analyzed above, chlorophyll contents were also positively correlated with K, Na, Ca, Mg and Fe, and we conjectured that the decrease of Mg and Fe was an important reason for the decrease of chlorophyll content. In other words, the decrease of Mg and Fe was an important reason for the decrease of biomass, which was consistent with previous results (Nazar et al., 2012).
In this study, Cd concentration in the shoot of ryegrass was positively correlated with K, Na, chlorophyll a and negatively correlated with Mn, MAD and CAT (Table S4 and Fig. 6). Previous studies had proved that addition of K as KCl or K2SO4 increased Cd concentrations in plant shoots of both Brookton and Krichauff (Zhao et al., 2004). But K and Na concentration in the shoot of ryegrass just had a slight decrease when C14 alkane concentration was in the range of 0.1–0.5% (Fig. 4a, b). In addition, Cd did not have a known biological function in plants and enters plant cells mainly via cation channels of Ca, Mg and Fe or transporters of other divalent cations (Huang et al., 2020). Therefore, we believed that the influence of K and Na on the Cd concentration in the shoot of ryegrass was limited.
Many researchers observed competitive uptake between Cd and Mn, Ca, Zn, Fe, because Cd and Mn, Ca, Zn, Fe use some common transporters for their uptake and translocation in plants (Rahman et al., 2016; Huang et al., 2020). As mentioned above, Mn concentration in the shoot of ryegrass significantly increased with the increase of C14 alkane concentration (Fig. 4f). Previous studies using distinct plant species evidenced that the increased shoot Mn accumulation reduced Cd uptake (Carvalho et al., 2019). In the presence of Cd, Peng et al. (2008) proved that adding Mn to the solution significantly reduced the concentrations of Cd in all organs of the plant. Thus, the increase of Mn in the shoots of ryegrass might be an important reason for the decrease of Cd concentration (Fig. 7).