Biological Effect of Gamma Rays According to Exposure Time on Germination and Plant Growth in Wheat
Biological Effect of Gamma Rays According to Exposure Time on Germination and Plant Growth in Wheat
Hong, Min Jeong;Kim, Dae Yeon;Jo, Yeong Deuk;Choi, Hong-Il;Ahn, Joon-Woo;Kwon, Soon-Jae;Kim, Sang Hoon;Seo, Yong Weon;Kim, Jin-Baek
2022-03-21 00:00:00
applied sciences Article Biological Effect of Gamma Rays According to Exposure Time on Germination and Plant Growth in Wheat 1 2 1 1 1 1 Min Jeong Hong , Dae Yeon Kim , Yeong Deuk Jo , Hong-Il Choi , Joon-Woo Ahn , Soon-Jae Kwon , 1 3 1 , Sang Hoon Kim , Yong Weon Seo and Jin-Baek Kim * Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu, Jeongeup 56212, Korea; hongmj@kaeri.re.kr (M.J.H.); jyd@kaeri.re.kr (Y.D.J.); hichoi@kaeri.re.kr (H.-I.C.); joon@kaeri.re.kr (J.-W.A.); soonjaekwon@kaeri.re.kr (S.-J.K.); shkim80@kaeri.re.kr (S.H.K.) Institute of Animal Molecular Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Korea; dykim@korea.ac.kr Division of Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Korea; seoag@korea.ac.kr * Correspondence: jbkim74@kaeri.re.kr Abstract: Gamma rays as a type of ionizing radiation constitute a physical mutagen that induces mutations and could be effectively used in plant breeding. To compare the effects of gamma and ionizing irradiation according to exposure time in common wheat (Keumgang, IT 213100), seeds were exposed to Co gamma rays at different dose rates. To evaluate the amount of free radical content, we used electron spin resonance spectroscopy. Significantly more free radicals were generated in the case of long-term compared with short-term gamma-ray exposure at the same dose of radiation. Under short-term exposure, shoot and root lengths were slightly reduced compared with those of the controls, whereas long-term exposure caused severe growth inhibition. The expression of antioxidant-related and DNA-repair-related genes was significantly decreased under long-term Citation: Hong, M.J.; Kim, D.Y.; Jo, gamma-ray exposure. Long-term exposure caused higher radiosensitivity than short-term exposure. Y.D.; Choi, H.-I.; Ahn, J.-W.; Kwon, The results of this study could help plant breeders select an effective mutagenic induction dose rate S.-J.; Kim, S.H.; Seo, Y.W.; Kim, J.-B. in wheat. Biological Effect of Gamma Rays According to Exposure Time on Keywords: wheat; gamma rays; exposure time; free radicals; growth inhibition; mutation breeding Germination and Plant Growth in Wheat. Appl. Sci. 2022, 12, 3208. https://doi.org/10.3390/ app12063208 1. Introduction Academic Editor: Richard Kouzes Wheat (Triticum aestivum L.) is one of the most important crops and accounts for Received: 9 February 2022 approximately 20% of the nutrient calories and proteins consumed by the global popula- Accepted: 19 March 2022 tion [1]. Wheat is an important source of essential or beneficial nutrients, such as proteins, Published: 21 March 2022 vitamins (notably several types of vitamin B), dietary fibers, and phytochemicals [2]. As a stable supply of wheat is required for food security due to the increase in the global Publisher’s Note: MDPI stays neutral population and climate change, a need for various genetic resources to support such a with regard to jurisdictional claims in published maps and institutional affil- supply is emerging. Plant breeders select resources suitable for their purpose by targeting iations. genetic resources with various traits to develop new varieties that can adapt to climate change response and consumption trends. In this process, the most important steps are creating genetic variation in and securing the genetic diversity of breeding materials. To increase genetic diversity, several researchers have used different techniques leading to Copyright: © 2022 by the authors. comprehensive changes in the plant genome, including traditional breeding techniques, Licensee MDPI, Basel, Switzerland. such as crossbreeding and mutagenesis, as well as new biotechnologies, such as genetic This article is an open access article transformation, genome editing, and introgression [3]. distributed under the terms and Ionizing radiation is most commonly used to generate useful mutations in plants due conditions of the Creative Commons to its ease of application and high mutation frequency. Ionizing radiation can cause direct Attribution (CC BY) license (https:// or indirect damage to plants. The direct effect causes damage to the genetic material (DNA creativecommons.org/licenses/by/ or RNA) in the cells of the organism, resulting in structural and functional changes in the 4.0/). Appl. Sci. 2022, 12, 3208. https://doi.org/10.3390/app12063208 https://www.mdpi.com/journal/applsci Appl. Sci. 2022, 12, 3208 2 of 14 DNA molecules [4,5]. Indirect damage generates highly reactive oxygen species (ROS), such as hydrogen peroxide (H O ), superoxide radicals (O ), and hydroxyl radicals (OH), 2 2 2 potentially leading to oxidative stress [6]. The reported effects of ionizing radiation on plant species include effects on the mutation rate, cytogenetic effects, and effects on biological responses [7–9]. Among the radiation sources, gamma rays are very important in mutation breeding and in vitro mutagenesis to the development of the required features in plants and increasing the genetic variability [10]. A previous report indicated that gamma rays affect changes in the phytochemical composition of plants [11]. Numerous studies on the use of gamma rays to develop new cultivars with diverse genetic backgrounds reported that gamma rays affected plant characteristics such as grain color, flower color, and plant height [12–16]. Approximately 63% of the mutant varieties registered in the Mutant Variety Database (a joint FAO/IAEA project) are varieties created through gamma irradiation [17]. Gamma irradiation is still considered an attractive tool for mutagenesis. Most previous research focused on the germination of seeds and physiological and biochemical changes in plants according to the dose of gamma radiation [18–20]. This study aimed to investigate the response in wheat according to different gamma radiation doses and exposure times. We irradiated wheat (Keumgang: a Korean wheat variety) seeds with 100, 300, and 500 Gy of gamma rays for 8 h and 14 days (the final dosages were the same, but the dose rates per hour were different after gamma irradiation). To compare the gamma irradiation effect according to the radiation dose and exposure time, we investigated oxidative stress, seed germination percentage, plant growth, chlorophyll content, antioxidant enzyme activity, and gene expression levels. Before gamma irradiation, we examined various doses and exposure times to determine which irradiation conditions could efficiently induce mutations. The results of this study could be used to select the appropriate gamma radiation dose to induce mutations in wheat mutation breeding. 2. Materials and Methods 2.1. Plant Material and Gamma Irradiation For gamma irradiation, seeds of Keumgang (National Agrobiodiversity Centre, RDA, Suwon, Korea; accession no. IT 213100), a wheat cultivar certified in Korea, were used. For the different gamma irradiation exposure times, we used two gamma irradiation facilities at the Advanced Radiation Technology Institute, an affiliate of the Korean Atomic Energy Research Institute (Jeongeup, Korea). A Co gamma phytotron (20 TBq of capacity, Nordion, ON, Canada) and a Co gamma irradiator (150 TBq of capacity, Nordion, ON, Canada) were used to irradiate seeds at low- and high-dose rates for 2 weeks and 8 h, respectively. The final exposure doses were set to 100, 300, and 500 Gy. The dose rates were 0.298 Gy/h, 0.893 Gy/h, and 1.488 Gy/h for the low-dose rates and 12.5 Gy/h, 37.5 Gy/h, and 62.5 Gy for the high-dose rates. After irradiation, the seeds were stored at 4 C until further analysis. 2.2. Germination Assays and Plant Growth To measure the germination percentage, 100 seeds were placed on a piece of moistened germination paper and then incubated at room temperature under a 16/8 h day/night cycle. The germinated seeds were counted each day for 5 days to determine the final germination percentage. Germination was considered complete when the seeds exhibited a radical emergence size of >2 mm. The germination percentage (GP), germination rate index (GRI), and mean germination time (MGT) were calculated using the following fomula: GP = n/N 100 (n = total number of seeds germinated; N = total number of seeds) [21]. The germination rate index was calculated according to the equation described by Esechie [22]: GRI = (N /i) (N = number of seeds germinated on day i). The MGT was calculated based i i on the formula described by Ellis and Roberts [23]: MGT = å(N T )/åN (N = number of i i i i seeds germinated, T = number of days from sowing). For plant growth, the seeds were germinated for 2 days at room temperature and then transferred into plastic containers containing Hoagland’s solution (Sigma, St. Louis, Appl. Sci. 2022, 12, 3208 3 of 14 MO, USA). The seedlings were grown in a phytotron room at 23 C with a photoperiod of 16/8 h (day/night). The Hoagland’s solution was exchanged each day. The shoot and root lengths were measured on day 10 after germination (DAG). DAG10 leaf samples were used for the measurement of chlorophyll content and antioxidant activities and the analysis of gene expression. 2.3. Chlorophyll Content Measurement The chlorophyll content was measured using a UV spectrophotometer as described by Lichtenthaler and Buschmann [24]. Briefly, wheat leaf samples (0.5 g) were homog- enized with 10 mL of 100% acetone and then kept overnight in the dark at 4 C. The homogenized sample mixtures were centrifuged at 10,000 rpm for 10 min and the su- pernatant was transferred into a new tube. The supernatant was used for pigment de- termination. The absorbance was measured using a UV-VIS spectrophotometer (Jenway, Chelmsford, UK) at 470, 644.8, and 661.6 nm. The chlorophyll contents were calculated as described previously [25]. The total chlorophyll, chlorophyll a, and chlorophyll b con- tents were calculated using the following equations: C = 11.24 A 2.04 A , a 661.6 644.8 C = 20.13 A 4.19 A , and C = 7.05 A + 18.09 A , respectively. 644.8 661.6 661.6 644.8 b a+b(total) 2.4. RNA Extraction and Gene Expression Analysis Total RNA of DAG10 leaves was extracted using Tri reagent (MRC, Cambridge, UK) according to the manufacturer ’s protocol. First-strand cDNA was prepared using a Power cDNA synthesis kit (iNtRON Biotechnology, Seongnam, Korea) according to the manufac- turer ’s protocol. RT-qPCR was carried out at a reaction volume of 20 microliters with SYBR premix Ex Taq II (Takara, Tokyo, Japan). Quantitative analysis was performed using the Bio-Rad CFX100 (Illumina, San Diego, CA, USA). All RT-qPCR amplification conditions were as follows: initial denaturation at 95 C for 5 min, followed by 40 cycles of 10 s at 95 C and 30 s at 65 C. The gene-specific primers are listed in Table S1. Actin (AB181991) was used as an endogenous control for the RT-qPCR analysis. 2.5. Free Radical Content Measurement Non-irradiated and irradiated freeze-dried and ground seed samples were transferred to a cylindrical quartz ESR glass tube (diameter, 5 mm). The glass tube was sealed from the open end using Whatman film (GE Healthcare, Buckinghamshire, UK). The glass tube was submitted for ESR measurements using an X band ESR spectrometer (JES-PX2300, JEOL, Tokyo, Japan) equipped with a cylindrical cavity. The operating conditions for the ESR measurements were as follows: power, 0.998 mW; microwave frequency, 9.429 GHz; modulation frequency, 100 kHz; modulation width, 1 mT; magnetic center field, 337.812 mT; sweep time 30 s; time constant, 0.03 s. The ESR signal intensities were assessed using the peak-to-peak amplitude of the first-derivative spectrum. 2.6. Total Phenolic Content The total phenolic content was measured using the Folin–Ciocalteu method with gallic acid (GA) as a standard [26]. In brief, 0.5 mL of wheat seed extract solution (methanol) was mixed with 2.5 mL of 10% Folin–Ciocalteu reagent. Next, the mixture was supplemented with 0.75 mL of 70% Na CO and incubated for 120 min at room temperature in the 2 3 dark. After incubation, the optical density was measured at 765 nm using a UV-VIS spectrophotometer (Jenway, Chelmsford, UK). The total phenolic content was calculated as mg/g of gallic acid equivalent (GAE) using the calibration curve of gallic acid. 2.7. DPPH Radical Scavenging Activity DPPH free radical scavenging capacity measurements were carried out using UV- VIS spectrophotometry [27]. The homogenized wheat seed samples were extracted using methanol for 24 h at 4 C. The extract solution (0.1 mL) was mixed with 3 mL of DPPH (0.1 mM) methanol solution and the absorbance was measured at 517 nm using a UV- Appl. Sci. 2022, 12, 3208 4 of 14 VIS spectrophotometer after reaction for 30 min at room temperature. The DPPH radical scavenging activity was calculated using the following equation: DPPH radical scavenging activity (%) = (1 A /A ) 100 sample control where A is the absorbance of the irradiated samples and A is the absorbance of sample control the control. 2.8. Antioxidant Enzyme Assay To determine the antioxidant enzyme activities, the protein content of the DAG10 leaves was extracted by grinding frozen samples with liquid nitrogen and homogenizing the samples in 1 mL of extraction buffer (0.2 M potassium phosphate buffer (pH 7.0) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA)) at 4 C. The total protein content was determined using the Bradford method [28]. The ascorbate peroxidase (APX) activity was determined using the Nakano and Asada method [29], and the catalase (CAT) activity was determined using the method of Abei [30]. The POD activity was measured following the method of Kwak [31] using pyrogallol as a substrate. The superoxide dismutase (SOD) activity was analyzed by monitoring the inhibition of the photochemical reduction of Nitro Blue Tetrazolium (NBT) according to the method of Giannopolitis and Ries [32]. 2.9. Statistical Analysis The statistical significance of the differences between the mean values was determined using one-way analysis of variance with Duncan’s multiple range tests. Significant differ- ences were evaluated at a 5% level of significance. All statistical analyses were performed using SPSS version 23 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Free Radical Contents and Plant Growth To examine the oxidative stress produced by gamma irradiation, we measured the free radical contents in irradiated and control seeds. The mean differences in the free radical levels were significant according to the gamma radiation dose and time (Figure 1). The free radical content increased dose-dependently in the case of both long- and short-term gamma irradiation. Interestingly, we observed that the free radical content was significantly higher in the long-term irradiated seeds compared to their short-term irradiated counterparts. The relative free radical levels significantly increased in the case of all long-term gamma irradiation treatments, showing a more than 5-fold increase compared with the control levels. In the case of the short-term treatment, the free radical content increased slightly (1.1–1.5-fold) compared with the control levels. Figure 2 shows the daily wheat seed germination percentage. The germination per- centages in DAG1 and DAG2 showed a significant difference between the short-term irradiation, including the control, and the long-term irradiation with gamma rays. The final germination percentage did not show a significant difference between the control and the irradiated seeds. Data analysis revealed that the GRI and MGT were affected by the gamma-ray dose and exposure time (Table 1). The GRI showed a tendency to decrease with prolonged exposure at the same dose of gamma radiation. The MGT was slower in the case of long-term irradiation. The MGT increased with an increase in the gamma-ray dose and exposure time. The shoot and root lengths decreased dose-dependently (Figure 3). The growth of plants treated with short-term gamma irradiation exposure was slightly reduced compared with the non-irradiated samples, whereas exposure to long-term irradiation caused severe growth inhibition. In the case of long-term irradiation, the shoot and root lengths decreased drastically as the radiation doses increased. These results indicate that the free radical content increase according to the radiation dose and exposure time increase is closely related to plant growth. Moreover, our results indicate that the high free radical levels affected the early stage of wheat development by reducing the ability of the seeds to germinate or delaying their development. The RD values determined by plant growth 50 Appl. Sci. 2022, 12, 3208 5 of 14 Appl. Sci. 2022, 12, x FOR PEER REVIEW 5 of 14 characteristics were 158.6 Gy and 206.3 Gy (shoot and root length in the case of short-term irradiation, respectively) and 202.8 Gy and 252.5 Gy (shoot and root length in the case of long-term irradiation, respectively) (Supplementary Figure S1). Figure 1. Measurements of the free radical content in wheat seeds following short- and long-term Figure 1. Measurements of the free radical content in wheat seeds following short- and long-term gamma irradiation using electron spin resonance (ESR). Con, non-irradiated seeds (0 Gy); S, short- gamma irradiation using electron spin resonance (ESR). Con, non-irradiated seeds (0 Gy); S, short- term irradiation; L, long-term irradiation; 100, 300, and 500, gamma irradiation doses. Each bar Appl. Sci. 2022, 12, x FOR PEER REVIEW 6 of 14 term irradiation; L, long-term irradiation; 100, 300, and 500, gamma irradiation doses. Each bar rep- represents the mean standard deviation (SD). Values with different letters are significantly different resents the mean ± standard deviation (SD). Values with different letters are significantly different using Duncan’s multiple range test (p < 0.05). using Duncan’s multiple range test (p < 0.05). Figure 2 shows the daily wheat seed germination percentage. The germination per- centages in DAG1 and DAG2 showed a significant difference between the short-term ir- radiation, including the control, and the long-term irradiation with gamma rays. The final germination percentage did not show a significant difference between the control and the irradiated seeds. Data analysis revealed that the GRI and MGT were affected by the gamma-ray dose and exposure time (Table 1). The GRI showed a tendency to decrease with prolonged exposure at the same dose of gamma radiation. The MGT was slower in the case of long-term irradiation. The MGT increased with an increase in the gamma-ray dose and exposure time. The shoot and root lengths decreased dose-dependently (Figure 3). The growth of plants treated with short-term gamma irradiation exposure was slightly reduced compared with the non-irradiated samples, whereas exposure to long-term irra- diation caused severe growth inhibition. In the case of long-term irradiation, the shoot and root lengths decreased drastically as the radiation doses increased. These results in- dicate that the free radical content increase according to the radiation dose and exposure time increase is closely related to plant growth. Moreover, our results indicate that the high free radical levels affected the early stage of wheat development by reducing the Figure 2. Gamma radiation dose and exposure time effects on seed germination percentage. The seed ability of the seeds to germinate or delaying their development. The RD50 values deter- germination percentages were determined daily for 4 days. DAG, days after germination. Each bar Figure 2. Gamma radiation dose and exposure time effects on seed germination percentage. The mi repr ne esents d bythe plmean ant g rowt SD. h characteristics were 158.6 Gy and 206.3 Gy (shoot and root length seed germination percentages were determined daily for 4 days. DAG, days after germination. Each in the case of short-term irradiation, respectively) and 202.8 Gy and 252.5 Gy (shoot and bar represents the mean ± SD. root length in the case of long-term irradiation, respectively) (Supplementary Figure S1). Table 1. Germination rate index and mean germination time according to the dose of and time of exposure to gamma radiation. Germination Rate Index (%) Mean Germination Time (Days) C 88.54 ± 1.97 1.295 ± 0.017 S-100 87.96 ± 3.42 1.320 ± 0.000 S-300 83.46 ± 0.14 1.425 ± 0.040 S-500 84.75 ± 4.23 1.390 ± 0.023 Figure 3. Gamma irradiation effect on plant growth under different doses and exposure times. (A) L-100 75.71 ± 0.91 1.600 ± 0.058 Image of plant growth under different irradiation conditions. Scale bar: 2 cm. (B) Shoot and (C) root L-300 69.88 ± 2.26 1.805 ± 0.040 length 10 days after germination. Each bar represents the mean ± SD (n = 10). Values with different letters L- a5 re 00 si gnificantly differen7 t u 1.0 sin 8 ± g Dunc 0.77an ’s multiple range test (p < 01.73 .05). 5 ± 0.029 3.2. Chlorophyll Content Determination The chlorophyll a content in wheat seedlings increased at 100 Gy (5.84 mg/g FW) and decreased at 300 Gy (5.39 mg/g FW) and 500 Gy (3.96 mg/g FW), and markedly decreased with long-term exposure compared with the control (Figure 4). Compared with the con- trol, 5.45 mg/g FW, the chlorophyll a content decreased by 38% at 500 Gy of short-term gamma irradiation. Furthermore, long-term gamma irradiation of 100 Gy (2.91 mg/g FW), 300 Gy (0.66 mg/g FW), and 500 Gy (0.73 mg/g FW) induced a significant reduction in chlorophyll content (by 47, 88, and 87%, respectively). We confirmed that the chlorophyll b and total chlorophyll contents showed similar patterns to the chlorophyll a content. These results indicate that 500 Gy of short-term gamma irradiation and long-term expo- sure to beyond 100 Gy of gamma radiation caused an obvious decrease in the chlorophyll a, chlorophyll b, and total chlorophyll contents in Keumgang. Appl. Sci. 2022, 12, x FOR PEER REVIEW 6 of 14 Appl. Sci. 2022, 12, 3208 6 of 14 Table 1. Germination rate index and mean germination time according to the dose of and time of exposure to gamma radiation. Germination Rate Index (%) Mean Germination Time (Days) C 88.54 1.97 1.295 0.017 S-100 87.96 3.42 1.320 0.000 S-300 83.46 0.14 1.425 0.040 S-500 84.75 4.23 1.390 0.023 L-100 75.71 0.91 1.600 0.058 Figure 2. Gamma radiation dose and exposure time effects on seed germination percentage. The L-300 69.88 2.26 1.805 0.040 seed germination percentages were determined daily for 4 days. DAG, days after germination. Each L-500 71.08 0.77 1.735 0.029 bar represents the mean ± SD. Figure 3. Gamma irradiation effect on plant growth under different doses and exposure times. Figure 3. Gamma irradiation effect on plant growth under different doses and exposure times. (A) (A) Image of plant growth under different irradiation conditions. Scale bar: 2 cm. (B) Shoot and Image of plant growth under different irradiation conditions. Scale bar: 2 cm. (B) Shoot and (C) root (C) root length 10 days after germination. Each bar represents the mean SD (n = 10). Values with length 10 days after germination. Each bar represents the mean ± SD (n = 10). Values with different different letters are significantly different using Duncan’s multiple range test (p < 0.05). letters are significantly different using Duncan’s multiple range test (p < 0.05). 3.2. Chlorophyll Content Determination The chlorophyll a content in wheat seedlings increased at 100 Gy (5.84 mg/g FW) and 3.2. Chlorophyll Content Determination decreased at 300 Gy (5.39 mg/g FW) and 500 Gy (3.96 mg/g FW), and markedly decreased The chloroph with long-termyll exposur a con e compar tent in wheat ed with theseed contrlings incre ol (Figure 4). Compar ased at ed 100 Gy with the (5.84 mg control, /g FW) and 5.45 mg/g FW, the chlorophyll a content decreased by 38% at 500 Gy of short-term gamma decreased at 300 Gy (5.39 mg/g FW) and 500 Gy (3.96 mg/g FW), and markedly decreased irradiation. Furthermore, long-term gamma irradiation of 100 Gy (2.91 mg/g FW), 300 Gy with long-term exposure compared with the control (Figure 4). Compared with the con- (0.66 mg/g FW), and 500 Gy (0.73 mg/g FW) induced a significant reduction in chlorophyll trol, 5.45 content mg/g FW, t (by 47, 88,h and e chlorophyl 87%, respectively). l a co W ntent d e confirmed ecre that ased theb chlor y 38 ophyll % atb 50 and 0 G total y of short-term chlorophyll contents showed similar patterns to the chlorophyll a content. These results gamma irradiation. Furthermore, long-term gamma irradiation of 100 Gy (2.91 mg/g FW), indicate that 500 Gy of short-term gamma irradiation and long-term exposure to beyond 300 Gy (0.66 mg/g FW), and 500 Gy (0.73 mg/g FW) induced a significant reduction in 100 Gy of gamma radiation caused an obvious decrease in the chlorophyll a, chlorophyll b, chlorophyl and total l cochlor ntent ophyll (by contents 47, 88, an in Keumgang. d 87%, respectively). We confirmed that the chlorophyll b and total chlorophyll contents showed similar patterns to the chlorophyll a content. These results indicate that 500 Gy of short-term gamma irradiation and long-term expo- sure to beyond 100 Gy of gamma radiation caused an obvious decrease in the chlorophyll a, chlorophyll b, and total chlorophyll contents in Keumgang. Appl. Sci. 2022, 12, x FOR PEER REVIEW 7 of 14 Appl. Appl. Sci. Sci. 2022 2022,, 12 12,, x FO 3208 R PEER REVIEW 7 o 7f of 14 14 Figure 4. Determination of total chlorophyll, chlorophyll a, and chlorophyll b contents upon gamma irradiation. Each bar represents the mean ± SD (n = 3). Values with different letters are significantly Figure 4. Determination of total chlorophyll, chlorophyll a, and chlorophyll b contents upon gamma different using Duncan’s multiple range test (p < 0.05). Figure 4. Determination of total chlorophyll, chlorophyll a, and chlorophyll b contents upon gamma irradiation. Each bar represents the mean SD (n = 3). Values with different letters are significantly irradiation. Each bar represents the mean ± SD (n = 3). Values with different letters are significantly dif diff fer eren ent t usi using ng Du Duncan’s ncan’s mu multiple ltiple range rangettest est ((p p < < 00.05). .05). 3.3. Gamma Radiation Effect on the Expression of DNA-Repair-Related Genes The effect of gamma irradiation on the expression of DNA-repair-related genes was 3.3. Gamma Radiation Effect on the Expression of DNA-Repair-Related Genes 3.3. Gamma Radiation Effect on the Expression of DNA-Repair-Related Genes analyzed using RT-qPCR (Figure 5). After gamma irradiation, most DNA-repair-related The effect of gamma irradiation on the expression of DNA-repair-related genes was an- The effect of gamma irradiation on the expression of DNA-repair-related genes was gene transcripts showed similar expression patterns, exhibiting a gradual decrease with alyzed using RT-qPCR (Figure 5). After gamma irradiation, most DNA-repair-related gene analyzed using RT-qPCR (Figure 5). After gamma irradiation, most DNA-repair-related increasing exposure doses. The expression of X-ray repair cross-complementing protein transcripts showed similar expression patterns, exhibiting a gradual decrease with increas- gene transcripts showed similar expression patterns, exhibiting a gradual decrease with (XRCC), KU70, and DNA methyltransferase 2 (DnMT2) was slightly upregulated by short- ing exposure doses. The expression of X-ray repair cross-complementing protein (XRCC), KU70, increasing exposure doses. The expression of X-ray repair cross-complementing protein term irradiation at 100 Gy (Figure 5A–C). The MSH6 transcript levels decreased with in- and DNA methyltransferase 2 (DnMT2) was slightly upregulated by short-term irradiation at (XRCC), KU70, and DNA methyltransferase 2 (DnMT2) was slightly upregulated by short- creasing gamma radiation doses (Figure 5D). The RT-qPCR results show that DNA-repair- 100 Gy (Figure 5A–C). The MSH6 transcript levels decreased with increasing gamma radia- term irradiation at 100 Gy (Figure 5A–C). The MSH6 transcript levels decreased with in- re tion latdoses ed gen (Figur e transc e 5D). ripts The were RT-qPCR drastica results lly re show duced that upo DNA-r n lon epair g-te-r rm elated irradi gene atio transcripts n compared creasing gamma radiation doses (Figure 5D). The RT-qPCR results show that DNA-repair- wit werh e drastically short-term redu irrad ced iatupon ion. T long-term hese results irradiation indicatecompar that long ed with -term short-term irradiation irradiation. negatively related gene transcripts were drastically reduced upon long-term irradiation compared im These pacts results the D indicate NA repair that long-term system in irradiation plants compared negatively witimpacts h short-the term DNA irrad repair iation system even at with short-term irradiation. These results indicate that long-term irradiation negatively in plants compared with short-term irradiation even at equal gamma-ray doses. equal gamma-ray doses. impacts the DNA repair system in plants compared with short-term irradiation even at equal gamma-ray doses. Figure 5. DNA-repair-related gene expression profiling in wheat seedlings determined by RT- Figure 5. DNA-repair-related gene expression profiling in wheat seedlings determined by RT- Figure 5. DNA-repair-related gene expression profiling in wheat seedlings determined by RT- qPCR qPCR. . ((A A)) XRCC, XRCC, (( B B)) KU KU70, 70, (C (C ) D ) DnMT2, nMT2, an and d (D (D ) MS ) MSH6. H6. RTR -q TPCR -qPCR was p waser performed formed witwith h three thr b ee io- qPCR. (A) XRCC, (B) KU70, (C) DnMT2, and (D) MSH6. RT-qPCR was performed with three bio- logical replicates, and each bar represents the mean ± SD (n = 10). Values with different letters are biological replicates, and each bar represents the mean SD (n = 10). Values with different letters are logical replicates, and each bar represents the mean ± SD (n = 10). Values with different letters are significantly different using Duncan’s multiple range test (p < 0.05). significantly different using Duncan’s multiple range test (p < 0.05). significantly different using Duncan’s multiple range test (p < 0.05). Appl. Sci. 2022, 12, x FOR PEER REVIEW 8 of 14 Appl. Sci. 2022, 12, 3208 8 of 14 3.4. Antioxidant-Related Gene Expression Levels To investigate how different gamma radiation exposure times affect the expression 3.4. Antioxidant-Related Gene Expression Levels of antioxidant-related genes, we performed RT-qPCR (Figure 6). APX, CAT, MnSOD, and To investigate how different gamma radiation exposure times affect the expression monodehydroascorbate reductase (MDHAR) showed similar gene expression patterns in of antioxidant-related genes, we performed RT-qPCR (Figure 6). APX, CAT, MnSOD, and plants subject to short-term irradiation (Figure 6A–C,H). CuZnSOD transcript levels de- monodehydroascorbate reductase (MDHAR) showed similar gene expression patterns in plants creased at 100 Gy of short-term irradiation and showed a similar level of expression to subject to short-term irradiation (Figure 6A–C,H). CuZnSOD transcript levels decreased that of the control at 300 Gy (Figure 6D). In the case of long-term irradiation, CuZnSOD at 100 Gy of short-term irradiation and showed a similar level of expression to that of the expression decreased with increasing gamma-ray doses. The glutathione reductase (GR) and control at 300 Gy (Figure 6D). In the case of long-term irradiation, CuZnSOD expression guaiacol peroxidase (GPX) gene expression levels were the highest when the plants were decreased with increasing gamma-ray doses. The glutathione reductase (GR) and guaiacol subject to short-term 100-Gy gamma irradiation, then decreased continuously with in- peroxidase (GPX) gene expression levels were the highest when the plants were subject to short-term 100-Gy gamma irradiation, then decreased continuously with increasing doses creasing doses (Figure 6E,F). Dehydroascorbate reductase (DHAR) transcripts were continu- (Figure 6E,F). Dehydroascorbate reductase (DHAR) transcripts were continuously induced ously induced up to 300 Gy, then decreased at 500 Gy of short-term irradiation (Figure up to 300 Gy, then decreased at 500 Gy of short-term irradiation (Figure 6G). Keumgang 6G). Keumgang subjected to long-term irradiation showed a significant decline in the ex- subjected to long-term irradiation showed a significant decline in the expression of most pression of most antioxidant-related genes. These results indicate that long-term irradia- antioxidant-related genes. These results indicate that long-term irradiation affects the tion affects the plants more seriously than short-term irradiation. plants more seriously than short-term irradiation. Figure 6. Antioxidant-related gene expression profiling in wheat seedlings determined by RT-qPCR. Figure 6. Antioxidant-related gene expression profiling in wheat seedlings determined by RT-qPCR. (A) APX, (B) CAT, (C) MnSOD, (D) CuZnSOD, (E) GR, (F) GPX, (G) DHAR, and (H) MDHAR. RT- (A) APX, (B) CAT, (C) MnSOD, (D) CuZnSOD, (E) GR, (F) GPX, (G) DHAR, and (H) MDHAR. qPCR was performed with three biological replicates, and each bar represents the mean ± SD (n = RT-qPCR was performed with three biological replicates, and each bar represents the mean SD 10). Values with different letters are significantly different using Duncan’s multiple range test (p < (n = 10). Values with different letters are significantly different using Duncan’s multiple range test 0.05). (p < 0.05). 3.5. Antioxidant Activity 3.5. Antioxidant Activity To estimate antioxidant enzyme activities upon different gamma radiation exposure To estimate antioxidant enzyme activities upon different gamma radiation exposure times, we measured the APX, CAT, POD, and SOD activities in wheat seedlings (Figure 7). times, we measured the APX, CAT, POD, and SOD activities in wheat seedlings (Figure APX activity was the highest at 100 Gy of long-term irradiation, and a lower value could be 7). APX activity was the highest at 100 Gy of long-term irradiation, and a lower value observed compared with the controls at 300 and 500 Gy of short- and long-term irradiation, could be observed compared with the controls at 300 and 500 Gy of short- and long-term respectively (Figure 7A). CAT activity levels were the highest at 300 Gy and the lowest irradiation, respectively (Figure 7A). CAT activity levels were the highest at 300 Gy and at 500 Gy of short- and long-term irradiation (Figure 7B). SOD activity was significantly the higher lowest in the at case 500 of Gy short-term of short-irradiation and longthan -term in ithe rradi contr atio ols, n (and Figure it decr 7B). eased SOD in the activi case ty was of long-term irradiation (Figure 7C). Upon short-term irradiation, the SOD showed the significantly higher in the case of short-term irradiation than in the controls, and it de- highest activity at 100 Gy and a tendency to decrease with increasing doses. POD activity creased in the case of long-term irradiation (Figure 7C). Upon short-term irradiation, the increased compared with the control after gamma irradiation, and POD activity levels were SOD showed the highest activity at 100 Gy and a tendency to decrease with increasing the highest in the case of long-term irradiation at 500 Gy (Figure 7D). doses. POD activity increased compared with the control after gamma irradiation, and POD activity levels were the highest in the case of long-term irradiation at 500 Gy (Figure 7D). Appl. Sci. 2022, 12, x FOR PEER REVIEW 9 of 14 Appl. Sci. 2022, 12, x FOR PEER REVIEW 9 of 14 Appl. Sci. 2022, 12, 3208 9 of 14 Figure 7. Short- and long-term gamma irradiation effects on (A) APX, (B) CAT, (C) SOD, and (D) POD antioxidant enzyme activities. Each bar represents the mean ± SD (n = 3). Values with different letters are significantly different using Duncan’s multiple range test (p < 0.05). Figure 7. Short- and long-term gamma irradiation effects on (A) APX, (B) CAT, (C) SOD, and (D) POD Figure 7. Short- and long-term gamma irradiation effects on (A) APX, (B) CAT, (C) SOD, and (D) antioxidant enzyme activities. Each bar represents the mean SD (n = 3). Values with different POD antioxidant enzyme activities. Each bar represents the mean ± SD (n = 3). Values with different 3.6. Total Phenolic Content and DPPH Free Radical Scavenging Activity letters are significantly different using Duncan’s multiple range test (p < 0.05). letters are significantly different using Duncan’s multiple range test (p < 0.05). The total phenolic content was 28.91 mg/g in the control (non-irradiated seeds). The 3.6. Total Phenolic Content and DPPH Free Radical Scavenging Activity 3.6. Total Phenolic Content and DPPH Free Radical Scavenging Activity total phenolic content slightly increased to 33.38 and 31.88 mg/g in the case of short-term The total phenolic content was 28.91 mg/g in the control (non-irradiated seeds). The irradiatio Tn he with total do phses enolic of 1 co 00 n tent and wa 50s 0 2G 8.91 y, re msg/g pectively in the con (Fig trure ol (n 8) on . -In irra the diacase ted s o eef dls) ong . Th -term e total phenolic content slightly increased to 33.38 and 31.88 mg/g in the case of short-term total phenolic content slightly increased to 33.38 and 31.88 mg/g in the case of short-term irradiation with doses of 100 and 500 Gy, respectively (Figure 8). In the case of long-term irradiation, a significant increase in the total phenolic content could be observed after irradiation with doses of 100 and 500 Gy, respectively (Figure 8). In the case of long-term irradiation, a significant increase in the total phenolic content could be observed after long-term gamma irradiation. The total phenolic content increased by 27.37%, 26.94%, and irrad long-term iation, gamma a signif irradiation. icant incThe rease total in phenolic the total content pheno incr lic eased conteby nt 27.37%, could b 26.94%, e observe andd after 23.48% compared with the control at 100-, 300-, and 500-Gy radiation doses, respectively. 23.48% compared with the control at 100-, 300-, and 500-Gy radiation doses, respectively. long-term gamma irradiation. The total phenolic content increased by 27.37%, 26.94%, and The DPPH free radical scavenging capacity decreased slightly but non-significantly after The DPPH free radical scavenging capacity decreased slightly but non-significantly after 23.48% compared with the control at 100-, 300-, and 500-Gy radiation doses, respectively. gamma irradiation. gamma irradiation. The DPPH free radical scavenging capacity decreased slightly but non-significantly after gamma irradiation. Figur Figure e 8. Diffe 8. Dif rfer ent ga ent gamma mma ra radiation diation dos dose and e an exposur d expo e su time re ef time fects ef onfect thes on (A) total the ( phenolic A) totacontent l phenolic con- Figure 8. Different gamma radiation dose and exposure time effects on the (A) total phenolic con- and (B) DPPH free radical scavenging activity. Each bar represents the mean SD (n = 3). Values tent and (B) DPPH free radical scavenging activity. Each bar represents the mean ± SD (n = 3). Val- tent and (B) DPPH free radical scavenging activity. Each bar represents the mean ± SD (n = 3). Val- with different letters are significantly different using Duncan’s multiple range test (p < 0.05). ues with different letters are significantly different using Duncan’s multiple range test (p < 0.05). ues with different letters are significantly different using Duncan’s multiple range test (p < 0.05). 4. Discussion 4. Discussion 4. Discussion Mutation breeding has been used as a tool to improve the genetic diversity of plants and develop desired traits. Gamma rays are a source of mutagens that can induce mu- Mutation breeding has been used as a tool to improve the genetic diversity of plants Mutation breeding has been used as a tool to improve the genetic diversity of plants tations in plants. Several breeders use gamma rays to select desired traits by securing and develop desired traits. Gamma rays are a source of mutagens that can induce muta- and develop desired traits. Gamma rays are a source of mutagens that can induce muta- genetic diversity. Various studies have shown the effects of gamma irradiation on seed tions in plants. Several breeders use gamma rays to select desired traits by securing ge- tions in plants. Several breeders use gamma rays to select desired traits by securing ge- germination, plant growth, chlorophyll content, oxidative stress, and secondary metabolite netic diversity. Various studies have shown the effects of gamma irradiation on seed ger- netic diversity. Various studies have shown the effects of gamma irradiation on seed ger- mination, plant growth, chlorophyll content, oxidative stress, and secondary metabolite mination, plant growth, chlorophyll content, oxidative stress, and secondary metabolite production [7,33]. In general, high-dose gamma irradiation is known to negatively influ- production [7,33]. In general, high-dose gamma irradiation is known to negatively influ- ence the physiological and biochemical traits of plants [9,34,35]. However, radiosensitivity ence the physiological and biochemical traits of plants [9,34,35]. However, radiosensitivity might reveal different levels of susceptibility depending on the plant variety due to DNA- might reveal different levels of susceptibility depending on the plant variety due to DNA- Appl. Sci. 2022, 12, 3208 10 of 14 production [7,33]. In general, high-dose gamma irradiation is known to negatively influence the physiological and biochemical traits of plants [9,34,35]. However, radiosensitivity might reveal different levels of susceptibility depending on the plant variety due to DNA-content-, repair-process-, antioxidant-reaction-, and cell cycle kinetics-related differences [36–39]. In order to increase the mutation breeding efficiency, the selection of an appropriate radiation dose that could increase the mutation frequency without significantly affecting the plant’s survival and reproduction is important. Most studies focused on the assessment of biologi- cal responses to different radiation doses, whereas relatively few studies have examined the different effects of gamma radiation exposure times [9,40,41]. Here, we report the main results of our study on the dose- and time-dependent effects of gamma irradiation, focusing on germination rate, plant growth, chlorophyll content, oxidative stress, gene expression, and antioxidant activity changes. Long-term exposure to gamma rays had a significantly negative effect on the seed germination percentage. We observed no differences in the final germination percentage between control and irradiated seeds. However, there was a significant difference in the germination pattern between short-term and long-term irradiation in the early germination stages (DAG1 and DAG2) (Figure 2). Germination-related indicators showed a tendency to decrease as the gamma radiation dose and exposure time increased (Table 1). Long-term gamma irradiation significantly reduced the GRI compared with short-term irradiation. We confirmed that the MGT increased as the exposure time increased, even at the same dose of gamma radiation (Table 1). Numerous studies have demonstrated that high gamma radiation doses cause physiological changes, such as a delay in seed germination and a reduction in the survival rate and plant growth, in wheat [19,20,42]. These results are consistent with those of previous researchers who reported that the seed germination potential decreased as the radiation dose increased. In addition, the germination speed was confirmed to be reduced with prolonged exposure to gamma rays even at the same radiation doses. Growth inhibition and retardation are known to be caused by cell cycle arrest in the G2 phase during somatic cell division upon exposure to high-dose gamma radiation [43,44]. Moreover, a gamma-radiation-mediated reduction in auxin and DNA synthesis could lead to a reduced mitotic frequency in the apical meristematic tissues, resulting in reduced plant growth and development [35,36,45]. This study on wheat’s response to gamma radiation showed that the gamma-radiation-related reduction in germination speed and plant growth could be attributed to the cell cycle arrest and changes in plant hormones and DNA synthesis. We found that the seed germination and seedling growth in wheat might be linked to the gamma radiation dose- and exposure time-related changes in free radical contents (Figures 1–3). Free radicals, such as ROS and reactive nitrogen species (RNS), are extremely reactive chemical compounds that potentially cause oxidative stress by damaging cell structures, including lipids, proteins, and DNA [46,47]. Free radicals play crucial roles in radiation sensitivity [48]. Previous studies have shown that high gamma radiation dose rates severely damage plants and induce the formation of more free radicals [49–51]. We evaluated the free radical levels according to the radiation dose and exposure time and observed that these levels increased linearly with increasing radiation doses in the case of both short- and long-term gamma irradiation (Figure 1). In particular, the number of free radicals was larger in the case of long-term irradiation than in the case of short-term irradiation. A previous study reported that chronically irradiated colored wheat exhibited lower free radical levels compared with the controls and showed that colored wheat anthocyanins could directly influence free radical scavenging capacity during the early developmental stages of wheat [52]. Unlike the results of previous studies, the results of this study show very high free radical levels in the wheat seeds subjected to long-term exposure to gamma radiation, and plant growth was also significantly reduced. Seeds that contain antioxidants (e.g., anthocyanins), such as those in colored wheat, even when exposed to gamma radiation for a long time, eliminate free radicals via antioxidants, thereby enabling plant growth. However, in the case of non-colored varieties of wheat, such as Keumgang, Appl. Sci. 2022, 12, 3208 11 of 14 the antioxidant activity is relatively low; thus, the free radical elimination ability is limited, which seems to negatively affect plant growth (Figure 3). These results suggest that when non-colored wheat seeds are continuously exposed to low doses of gamma radiation, the free radical production increases and this increase in free radicals induces oxidative stress, negatively affecting wheat seed germination and seedling growth. Chlorophyll content is a useful indicator for evaluating the physiological response to radiation [53]. A previous report indicated that exposure to 100 Gy of gamma radiation increased the content of chlorophyll in wheat [18]. In this study, short-term irradiation at 100 Gy produced the same result (Figure 4). Our results indicate that high gamma radiation doses decreased the chlorophyll content in comparison with the control. In particular, the chlorophyll content decreased significantly in the case of longer gamma radiation exposure times (Figure 4). Numerous studies have reported that high gamma radiation doses reduce photosynthetic activity, ultimately decreasing the chlorophyll content and plant growth [34,41]. This result is consistent with those of previous studies reporting that plants exposed to gamma radiation exhibited lower chlorophyll contents than the controls. We also confirmed that the chlorophyll content in the wheat seeds exposed to gamma radiation significantly decreased in the case of long-term compared with short-term irradiation. Gamma radiation can affect the integrity of genetic information and impair genomic stability by inducing DNA damage. In contrast to animals, plants are constantly exposed to the threat of DNA damage as they cannot change locations to avoid unfavorable growth conditions. Under adverse conditions, most organisms have mechanisms, such as DNA repair, that prevent mutations caused by damaging ionizing radiation, and the DNA strand breaks induced by ionizing radiation can be repaired with fast kinetics. Ptácek et al. [54] found that the DNA damage induced by 30 Gy of gamma radiation in tobacco was perfectly repaired within 24 h. The DNA-repair-related genes allow for the repair of ionizing- radiation-induced damage. Previous reports indicated that the DNA-repair-related genes, such as PnLIG4, PnKU70, PnXRCC4, PnPCNA, and PnRAD51, from Lombardy poplar (Populus nigra) were upregulated upon gamma irradiation [55]. In addition, the AtRAD51, AtLIG4, and AtXRCC4 transcripts were upregulated upon gamma irradiation treatment [56]. We assessed DNA-repair-related gene expression patterns at various gamma radiation exposure times and doses (Figure 5). We observed that the expression of most DNA-repair- related genes (XRCC, KU70, DnMT2, and MSH6) was downregulated by gamma radiation in a dose- and time-dependent manner except for the case of short-term irradiation at 100 Gy. DNA-repair-related gene transcript levels in the case of long-term irradiation were lower than in the case of short-term irradiation. We demonstrated that long-term exposure to gamma radiation affected wheat growth more seriously than short-term exposure through DNA-repair-related gene expression levels. To avoid gamma-radiation-induced oxidative stress, plants use antioxidant defense systems comprised of enzymatic antioxidants (such as APX, CAT, SOD, GR, GPX, MDHAR, and DHAR) and non-enzymatic antioxidants (such as ascorbic acid (AA), reduced glu- tathione (GSH), -tocopherol, carotenoids, and flavonoids) [47]. The antioxidant defense systems are used to maintain intracellular homeostasis by regulating cellular ROS levels, resulting in the protection of plants against various stress conditions [57]. To determine the antioxidant activity in wheat plants at different gamma radiation doses and expo- sure times, antioxidant-related gene expressions and antioxidant enzyme activities were measured (Figures 6 and 7). We found that antioxidant-related gene transcripts showed lower expression levels in the case of long-term irradiation compared with the control and the case of short-term-irradiation. The antioxidant enzyme activities did not show the same pattern as the antioxidant-related gene expressions. This result indicates that the gamma-radiation-generated excess ROSs were not eliminated to an appropriate extent due to the reduced gene expression and antioxidant enzyme activity, thereby negatively affecting wheat plant growth. In this study, the SOD, CAT, and APX gene transcript profiles displayed no direct correlation with the corresponding enzymatic activities, potentially due to the existence Appl. Sci. 2022, 12, 3208 12 of 14 of several different antioxidant enzyme isoforms [58]. In this study, no direct relationship appeared to exist between gene expression and antioxidant enzyme activity. However, elucidating the antioxidant-related gene expression patterns could help us to better under- stand the molecular mechanisms underlying how different gamma radiation doses and exposure times affect wheat. As most investigations on the effects of gamma irradiation were performed under short-term irradiation conditions, the findings of this study provide direct information on various effects of gamma irradiation on wheat mutagenesis through the comparison of short- and long-term exposure to gamma radiation. Based on previous research, 100 Gy of gamma radiation can be considered an optimal dose for mutagenesis, and the effective dose for generating a high degree of genetic diversity and a small amount of physical damage is 200–400 Gy. Furthermore, 200 Gy of gamma radiation is an appropriate dose to obtain a drought-resistant wheat mutant line [59–61]. However, the optimal dose of gamma radiation for mutagenesis differs depending on the cultivar or desired agricultural trait. The results of our study investigating the gamma irradiation conditions-related correlation between plant oxidative stress and plant growth response in non-colored wheat provide useful information for the gamma-radiation-assisted mutation breeding of wheat. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/app12063208/s1, Figure S1: RD50 values in response to gamma- rays for growth characteristics of Keumgang, Table S1: Primers used for gene expression analysis. Author Contributions: Conceptualization, M.J.H. and J.-B.K.; formal analysis, M.J.H. and D.Y.K.; methodology, M.J.H., S.H.K. and D.Y.K.; data curation, M.J.H. and D.Y.K.; writing—original draft preparation, M.J.H.; writing—review and editing, Y.D.J., H.-I.C., J.-W.A., S.H.K., S.-J.K. and Y.W.S.; supervision, J.-B.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by grants from the Nuclear R&D Program of the Ministry of Science and ICT (MSIT) and the research program of KAERI, Republic of Korea. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. 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