Heritable epigenetic modifications of plants generally occur in response to environmental variation, further altering phenotypes [10–13]. As the first discovered and intensely studied epigenetic regulatory mechanism, DNA methylation plays a vital part in regulating plant growth [10, 11, 15]. Therefore, We measured DNA methylation levels after La(III) exposure (Fig. 1). We found that short-term (0.5 d) La(III) exposure did not affect DNA methylation levels in underground and aboveground parts of Arabidopsis thaliana seedlings. With increased exposure time, low-dose (30 µM) La(III) exposure for 1.0 d increased DNA methylation levels in underground parts. When the exposure time was 2.0–8.0 d, DNA methylation levels in underground and aboveground parts rose. In contrast to the exposure to low-dose La(III), exposure to high-dose (80 µM) La(III) for 1.0 d did not change the plants’ DNA methylation in underground parts. Under high-dose La(III) exposure for 2.0–8.0 d, DNA methylation levels in underground parts first increased (2.0 d) and then decreased (4.0 and 8.0 d), and DNA methylation levels in aboveground parts only decreased over time. The effect of the duration of La(III) exposure on the level of DNA methylation suggests that when conducting REEs pollution screenings and remediation, we should select several plants as sentinels and the level of DNA methylation can be used as a marker to evaluate the remediation effect of REE pollution and whether REEs exist in the environment.
The above results raised the question of how exogenous La(III) changes the level of DNA methylation. SAM produced in the process of one-carbon metabolism acts as a direct donor of methyl groups for DNA methylation. In addition, SAM is demethylated into SAH by the DNA methyltransferase [30–33]. A large number of studies have shown that the imbalance of one-carbon metabolites can affect DNA methylation [34], and the SAM/SAH is regarded as an index of methylation potential [35, 36]. In this study, we measured the contents of SAM and SAH after La(III) exposure, calculated the SAM/SAH, and analyzed the relationship between the three indices and the DNA methylation levels by correlation analysis (Fig. 2; Table 1). Based on the results, we speculate that short-term La(III) exposure has no effect on DNA methylation levels, and plant resistance to environmental variation plays a key role in that suppressed the changes in one-carbon metabolism, inducing DNA methylation levels to stay the same status. With extended exposure time, low-dose La(III) exposure reduced one-carbon metabolism, thereby inducing an increase in DNA methylation levels. In contrast, high-dose La(III) exposure increased one-carbon metabolism, thereby inducing a decrease in DNA methylation levels [except La(III) exposure for 2.0 d]. There are two possible reasons for the above phenomena. First, low-dose La(III) exposure might promote the increase of methyltransferase activity, thereby a large amount of SAM would be demethylated to form SAH. In contrast, high-dose La(III) exposure might have inhibited the methyltransferase activity and thereby reduced the number of methyl groups and inhibiting DNA methylation [37]. Second, low-dose La(III) exposure might have reduced the SAM content by reducing the activity of the methionine adenosyl transferase, which is known to show enhanced activity due to SAM accumulation [38]. The resulting reduction of methyl groups would ultimately lead to decrease DNA methylation levels. Consistently, high-dose La(III) exposure would greatly increase the SAM content by enhancing the activity of adenosyl transferase. In addition, we found that when plants were exposed to high-dose La(III) for 2.0 d, one-carbon metabolism increased, together with the DNA methylation levels in underground parts. On the one hand, after La(III) exposure for 1.0 d, one-carbon metabolism provided many methyl groups for DNA methylation. These methyl groups were sufficient for methyltransferase utilization after La(III) exposure for 2.0 d, thus increasing the level of DNA methylation in underground parts. On the other hand, at 2.0 d of La(III) exposure, the high DNA methylation levels in underground parts was negative for the one-carbon metabolism, reducing its methyl supply, which increased the level of DNA methylation in underground parts.
Based on the above research, we conclude that exogenous La(III) affects the level of DNA methylation of Arabidopsis thaliana by regulating one-carbon metabolism. So, how exogenous La(III) affect the growth of Arabidopsis thaliana by changing the level of DNA methylation? We measured the growth indices of Arabidopsis thaliana and explored the relationship between DNA methylation levels and growth using pharmacological methods and correlation analyses (Figs. 3–6; Table 2). The results showed that short-term La(III) exposure did not affect the growth of Arabidopsis thaliana. We speculate that the plants have the ability to resist brief La(III) stress [39, 40] and maintain a stable DNA methylation levels, so there is no change in growth indices. In addition, studies have shown that changes in soil bacterial communities may also lead to changes in plant growth [41]; we speculate that short-term La(III) exposure did not change the Arabidopsis root bacterial community and thus did not change its growth. With increased exposure time, low-dose La(III) exposure for 1.0 d increased DNA methylation levels in underground parts, followed by increased primary root length and root hair number. When the exposure time was 2.0–8.0 d, DNA methylation levels in underground and aboveground parts rose, and the plant heights (2.0 d), lateral root numbers (2.0 d), leaf areas (4.0 d), and leaf numbers (8.0 d) also increased. We speculate that the main reasons for the above-mentioned changes in growth after exposure to low-dose La(III) are as follows: on the one hand, the changes in DNA methylation levels after La(III) exposure affect plant photosynthesis [42]. The increased DNA methylation enhanced the net photosynthetic rate of the plants, provided sufficient material and energy for the growth and development, and increased the plants’ growth indices. On the other hand, DNA methylation can regulate gene expression and thereby influence the abundance of trait-specific genes. For example, the reduction of plant height, leaf number, leaf area, primary root length, lateral root number, and root hair number is due to the overexpression of various genes, including 5PTase (At1g05470) [43, 44], CRK28 (At4g21400) [45, 46], and SCN1 (At3g07880) [47]. Thus, low-dose La(III) might increase DNA methylation levels, inhibit the expression of these genes, ultimately leading to the observed changes in the growth indices. In contrast to the exposure to low-dose La(III), exposure to high-dose La(III) for 1.0 d did not change the plants’ DNA methylation in underground parts. Still, the primary root length and lateral root number increased, and the root hair number decreased in these plants. We speculate that when plants were exposed to high-dose La(III) for only 1.0 d, the treatment might not have caused changes in DNA methylation levels of the whole genome, it might have caused methylation of promoter regions. Methylation of the DNA promoter region has a strong inhibitory effect on gene expression [48]; thus, it could have inhibited the expression of genes regulating primary root length and lateral root number, ultimately promoting the elongation of the primary root and the growth of lateral roots [45, 46]. Under high-dose La(III) exposure for 2.0–8.0 d, DNA methylation levels in underground parts first increased (2.0 d) and then decreased (4.0 and 8.0 d), and DNA methylation levels in aboveground parts only decreased over time. Simultaneously, the plant height, leaf area, and primary root length decreased at 4.0 and 8.0 d and leaf number decreased at 8.0 d, while the lateral root number increased, and the root hair number decreased. We speculate that the main reason for the above phenomena is that DNA methylation levels in underground and aboveground parts increased and decreased simultaneously under high-dose La(III) exposure for 2.0 d, which jointly regulated the plant height and primary root length, and finally left them unchanged. When DNA methylation levels in aboveground parts decreased, they might have decreased the expression level of 5PTase [43, 44], which could not change the leaf number and leaf area. In addition, the increase of DNA methylation levels in underground parts may have increased the lateral root number by reducing the expression of CRK28 [45, 46], or it might have increased the lateral root number and decreased the root hair number by changing the distribution of auxin in roots [6]. When the exposure to high-dose La(III) was prolonged to 8.0 d, DNA methylation levels in underground and aboveground parts decreased, potentially causing the overexpression of 5PTase [43, 44], CRK28 [45, 46], and SCN1 [47], thereby reducing plant height, leaf number (8.0 d), leaf area, primary root length, and root hair number. Simultaneously, the auxin contents might have been reduced in primary roots and increased in lateral roots, which could have further promoted the growth of lateral roots [6]. In addition, the reduction of DNA methylation levels could weaken chromosome stability, make chromosomes abnormal, and then inhibit cell division and plant growth [49, 50].
The above studies have found that La(III) exposure had a dose-response and time-response, that is, short-term La(III) exposure had no effect on one-carbon metabolism and DNA methylation levels, so it had no effect on the growth. Long-term and low-dose La(III) exposure reduced one-carbon metabolism and increased DNA methylation levels, thereby promoting growth, long-term and high-dose La(III) exposure was opposite, ultimately reducing plant height, leaf number, leaf area, primary root length and root hair number, increase lateral root number (Fig. 7).