Using Relaxation Time to characterize biological effects of different mutagens

All kinds of mutagenic factors may cause physiological, biochemical and genetic changes of all organisms. To characterize their characteristic biology effects, the concept of Relaxation Time (RT) was introduced for the first time, and the specific process was as follows. After mutation of organisms, the offsprings will be continuingly cultured (or cultivated) to the next generation (Rx). Once a biological effect began to show no significant difference compared to the untreated controls, the Rx was defined as the RT of the effect. In this paper, three kinds of mutagenic factors were selected to treat the seeds or seedlings of Astragalus sinicus L., subsequently, the corresponding RT was calibrated. The results showed that the RT was diverse not only among different biological effects but also among different mutagenic factors. For the RT of chemical mutagens and gamma rays, most of which are concentrated on R1, whereas the heavy ion beams have significant differences among different tracks. Among biological effects, the SOD activity and superoxide anion free radical content in the Peak region are more prominent, and their RT reaches R3 and R4, respectively. Thus, the RT may characterize the characteristic biological effects from differently mutagenic factors.


Results
Overview. All raw data were lined in the Supplementary Materials (Additional information) "S-RD 1", "S-RD 2", and "S-RD 3". Starting from the raw data, compared with the respective control, the difference of germination potential (GP 1 ) or germination percentage (GP 2 ) was more than 15% and the difference of SOD activity, superoxide anion free radical content (SAFAC) or biomass per plant (BPP) reached a level of p ≤ 0.05, which was listed as a significant difference, and the statistical results were shown in Figs. 1, 2, 3, 4, 5, 6, 7, 8 and 9 as follows.
Gamma-ray trials (Fig. 1). Compared with the control, only higher doses of the germination potential (GP 1 ) and the germination percentage (GP 2 ) had more than 15% differences in R 0 (i.e., RT = R 1 ), while other generations (i.e., RT = R 0 ) had no significant difference. For the SOD activity, R 0 (i.e., RT = R 1 ) at all doses and R 1 (i.e., RT = R 2 ) at high doses showed significant differences compared with controls (p = 0.05). For the superoxide anion free radical content (SAFRC ) at all doses and the biomass per plant (BPP) at high doses, only there was a   www.nature.com/scientificreports/ significant difference in R 0 (i.e., RT = R 0 ) compared with the control, while there was no significant difference in the BPP at low dose (i.e., RT = R 0 ). The analysis results were listed in Table 1.
Heavy ion beam trials (Figs. 2, 3, 4, 5, 6, 7). Heavy ion beam irradiation is a complex process: six Petri dishes with seeds were stacked along 80 meV/u carbon ion beam tracks and separated into six regions to test after irradiation (see "Methods" for details), respectively. Petri dish 1 and 2, were equivalent to the Plateau regions of the Bragg curve; Petri dish 3 was close to the Peak of the Bragg curve; Petri dish 4 was the Peak-Tail junction region; and Petri dish 5 and 6 were the Tail regions of irradiation. Figure 2 showed the results of Petri dish 1. Compared with the control, only R 0 (i.e., RT = R 1 ) of the GP 1 and the GP 2 had more than 15% differences at higher doses, while other generations (i.e., RT = R 0 ) had no significant differences. There were significant differences in R 0 and R 1 (i.e., RT = R 2 ) in the SOD activity compared to control (p ≤ 0.05). There were significant differences in R 0 , R 1 and R 2 (i.e., RT = R 3 ) of the SAFRC (p ≤ 0.05). For the BPP, there was significant difference in R 0 (i.e., RT = R 1 ) at high doses compared with the respective control. Figure 3 showed the results of Petri dish 2. Only the GP 1 and the GP 2 were measured. Compared with the control, only R 0 generations (i.e., RT = R 1 ) of the GP 1 and the GP 2 had more than 15% differences.   www.nature.com/scientificreports/ Figure 4 showed the results of Petri dish 3. For the GP 1 and the GP 2 , only R 0 (i.e., RT = R 1 ) had more than 15% differences compared with the control. R 0 , R 1 , and R 2 (i.e., RT = R 3 ) of the SOD activity at all doses, R 0 , R 1 , R 2 and R 3 (i.e., RT = R 4 ) of the SAFRC at all doses, and R 0 , R 1 (i.e., RT = R 2 ) of the BPP at high doses showed significant differences compared to controls (p ≤ 0.05).   www.nature.com/scientificreports/ Figure 5 showed the results of Petri dish 4. Only the GP 1 and the GP 2 were also measured. Compared with the control, R 0 (i.e., RT = R 1 ) of the GP 1 at all doses and R 0 (i.e., RT = R 1 ) of the GP 2 at high doses had more than 15% differences. Figure 6 showed the results of Petri dish 5. Compared with the control, there was no generations with more than 15% differences in the GP 1 (i.e., RT = R 0 ) and the GP 2 (i.e., RT = R 0 ). The results of the SOD activity was complicated: there were significant differences in R 0 and R 1 , while the R 3 or the R 4 at a dose also showed significant differences (p ≤ 0.05). After induction, we finalized the RT of the SOD activity as R 2 . For the SAFRC, the significant differences were observed in R 0 , R 1 (i.e., RT = R 2 ) at the highest dose and R 0 (i.e., RT = R 1 ) at other doses compared with the control (p ≤ 0.05). For the BPP, there is no significant differences in all generations (i.e., RT = R 0 ) compared with the control (p ≤ 0.05). Figure 7 showed the results of Petri dish 6. Only the GP 1 and the GP 2 were also measured. There were no more than 15% different generations compared with the respective control, thus, the RT of both the GP 1 and the GP 2 were R 0 . The results of the analysis in the Region 1-6 from heavy ion beam irradiation were also listed in Table 1. DES processing trials (Fig. 8). Because the plantlets with normal germination were selected for chemical mutagens treatment, there was no R 0 determination of the GP 1 and the GP 2 (see "Methods" for details). Compared with the control, there were no generations (i.e., RT = R 1 ) with more than 15% difference in the GP 1 and the GP 2 . For the SOD activity, the SAFRC and the BPP, compared with the respective controls, there were only significant differences in R 0 (i.e., RT = R 1 ) at high doses, whereas other treated generations (i.e., RT = R 0 ) had no significant differences (p ≤ 0.05) at low doses. The analysis results were also listed in Table 1. MH processing trials (Fig. 9). MH was completely consistent with DES processing results. The analysis results were also listed in Table 1.
RT analysis at seed germination stage. Using the RT of GP 1 or GP 2 may characterize this performance (Table 1 as shown). The GP 1 may reflect the uniformity of seed germination, whereas the GP 2 may reflect the survival ability of the seeds and the intensity of the action of the various mutagenic factors to the seeds. Table 1 indicated that the variation trend of the RTs of both the GP 1 and the GP 2 was consistent, compared with the untreated control group. For the irradiation in the gamma-rays and the Plateau regions and the Tail regions along carbon ion beam pathway at low doses, the RTs of the GP 1 and the GP 2 were R 0 , whereas the RTs of the GP 1 and the GP 2 of the treatments from two chemical mutagens and the Peak regions along carbon ion beam pathway were R 1 .
RT analysis at seedling stage. Using the RT of SOD activity or SAFRC may characterize their performances (Table 1 as shown). The SOD activity may reflect the cell stress facing to the mutagenic factors, while the SAFRC cannot only reflect the stress of the cells, but also reveals the indirect effects of various mutagenic factors, the results are shown in Table 1. It showed that the RT of SODA was R 1 after the treatments of low dose gamma rays, the Tail region along carbon ion beam pathway and two chemical mutagens, compared with untreated control group; the RT of the Peak region along carbon ion beam pathway was R 3 ; and the RT of the other treatment group was R 2 . The treatment of gamma-ray, low-dose Tail region along carbon ion beam pathways and two chemical mutagens were performed, the RT of the SAFRC was R 1 ; the RT of the SAFRC the Plateau region and the Peak region along carbon ion beam pathway were R 3 and R 4 , respectively. www.nature.com/scientificreports/ www.nature.com/scientificreports/ RT analysis at plant maturation stage. The performance of biomass per plant was characterized by the RT (Table 1 as shown). The BPP cannot only reflect the growth of Astragalus sinicus L. plants, but also reflect the synthesis and accumulation of plant cell metabolites. Table 1 revealed that the RT of the BPP of gamma-ray, the Plateau region and the Tail region along carbon ion beam pathway as well as chemical mutagens was mainly R 0 or R 1 , compared with the untreated control group, whereas the RT of the BPP of the Peak region at high-doses along carbon ion beam pathway was R 2 .

Discussion
In general, the RT is significantly different not only among different mutagenic factors and but also among different biological effects. The RT of gamma rays and chemical mutagens is relatively short. After planting a generation, except for mutants, the population affected by them can basically restore homeostasis. Whereas the results of the action of the heavy ion beams are the most complex, and the RT distribution of its biological effects is from R 0 to R 4 , which may be related to the existence of a variety of physicochemical mechanisms such as energy deposition, mass deposition, charge effects, and momentum transfer along different tracks of the heavy ion beams 9,19 , especially in the treatment of the Peak region, some biological effects requires a longer time to restore the population homeostasis. The longer RT after irradiation by heavy ion beams may reflect the accumulation of DNA mutations and chromosome rearrangements. Generally, heavy ion beams are more mutagenic than gamma rays, diethyl sulfate, or maleyl hydrazine [23][24][25] . According to the RT of different biological effects, it has positive application value in at least three aspects. First, during the breeding of mutagenesis mutants, the different strategy can be adopted: generally speaking, because of the longer stable time on the population, the biological trait with long Relaxation Time needs more generations to choose and accumulate useful individual plants. Second, in the study of stress and adaptability of biological individuals, it can be distinguished based on the length of Relaxation Time: the biological effects of short RT are stress traits, while the biological effects of long RT are adaptive traits. The third is that, in the biological study of phylogeny, because the organisms have experienced the action of natural environment factors for a long time, seeking and using some primitive or starting population as the control, the concept of the RT can still be introduced. By comparing the RT difference of specific biological effects among populations, the way of some population evolution can be speculated. Especially in the era of bioomics popularity, from the gene expression and metabolic pathway changes to metabolic network connectivity, we can quickly obtain the comparison results of related indicators such as nucleotide sequences between different species or individuals, which can be easily characterized by their RTs.
Not only is the biology a complex system [16][17][18] , but its mutation is also a complex processing. as a result, there will be a rebalancing procedure among differently biological macromolecules (such as DNA, RNA, proteins and chromosome), different organelles (such as mitochondrion) and different metabolic pathways (such as antioxidant systems regulated by ROS). Thus, fully revealing the RTs of those life processes and inherited characters will be our important research objectives. , and the six Petri dishes were stacked to be used in irradiation test with 80 meV/u carbon ion beams on the TR 4 terminal of the Lanzhou Heavy Ion Accelerator (Fig. 10). According to the theoretical calculation (Lise ++ 9.9: https ://lise.nscl.msu.edu/lise.html) and the measurement of CR-39 heavy ion track detectors 23,24 , when 87.5 meV/u of carbon ion beams was used to test, the Bragg peak of the energy loss appears near 2.2 cm depth in water and between 2.8 and 3.4 cm depth in Arabidopsis seeds along the tracks (Wang et al. 25 ). While 80 meV/u of carbon ion beams was used to this test, the Bragg peak of the energy loss appears between 2.2 and 3.2 cm depth (not spread-out Bragg peak) in Astragalus sinicus L. seeds along the tracks (about at the Petri dish 3). After irradiation, the whole installation was divided into six Petri dishes, which were classified as Petri dish 1-6 from close to far away according to the contact of carbon ion beams: Petri dish 1 of the Plateau region 1, Petri dish 2 of the Plateau region 2, Petri dish 3 of the Peak region, Petri dish 4 of the Peak-tail connect region, Petri dish 5 of the tail region 1 and Petri dish 6 of the tail region 2. The seeds of each Petri dish were applied to subsequent experiments.

Methods
Treatment of chemical mutagens. The whole seedlings of Astragalus sinicus L. (at 1-2 leaf stage) were treated with different concentrations (0-1.3%) of diethyl sulfate (DES) and maleyl hydrazine (MH) solutions at room temperature for 24 h, respectively. After washing with the sterilization water, their survival curves were plotted by statistics on survival ratios (as shown in Fig. 11). According to Fig. 11, the working concentrations of DES and MH of 0, 0.1%, 0.3% and 0.5% were used to test the seedlings. In 2015, the seeds of Astragalus sinicus L. harvested in 2014 were germinated. After about 10 days (at 1-2 leaf stage), the above working concentrations of DES and MH solution were sprayed to the whole seedlings of R 0 generation for 24 h, respectively. After washing Scientific RepoRtS | (2020) 10:13941 | https://doi.org/10.1038/s41598-020-70600-2 www.nature.com/scientificreports/ with the sterilization water, we sampled and carried out cultivation successions according to different treatments for subsequent trials. Just because of this kind of treatment, the germination potential and germination percentage of R 0 generation were not counted in this paper.

Germination test.
A Petri dish (a diameter of 150 mm and depth of 25 mm) with seeds was used for the germination test at room temperature and natural light. The germination potential (GP 1 ) was counted by the number of Astragalus sinicus L. germination within 7 days, and the germination percentage (GP 2 ) was counted by the number of all germination seeds about 20 days before transplanting. The chemical mutagen treatment group performed germination potential and germination percentage statistics from the R 1 generation seeds (i.e., the seeds harvested by the R 0 -generation plants), while the other treatment groups performed germination potential and germination percentage statistics from the R 0 generation (the starting seeds harvested in 2014).
Determination of SOD activity and Superoxide anion free radical content. At the later stage of the germination test (about 20 days, including seedlings with chemical inducer treatment), the whole plant of 20 seedlings was randomly selected from each dish, and the crude extract of seedlings was extracted according to Xiao HS, et al. 26 and stored in a refrigerator at -20℃ for the determination of SOD activity (SODA) and Superoxide anion free radical content (SAFRC), which the determination process was carried out according to the method from Xiao HS, et al. 26 and Simova-Stoilova LK, et al. 27 .
Field trials and determination of biomass per plant. All seedlings were transplanted into the field after about 20 days of indoor germination (including chemical mutagen-treated seedlings). three repeated, random block design with a plot of 12 m 2 , and the plant spacing was 30 cm (North-South) × 20 cm (East-West) to ensure that each individual plant can grow normally and was rarely affected by other plants. Up to maturity,