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Jung et al. BMC Plant Biology (2019) 19:561 https://doi.org/10.1186/s12870-019-2056-8 RESEARCH ARTICLE Open Access Overexpression of rice jacalin-related mannose-binding lectin (OsJAC1) enhances resistance to ionizing radiation in Arabidopsis In Jung Jung1†, Joon-Woo Ahn1†, Sera Jung1, Jung Eun Hwang2, Min Jeong Hong1, Hong-Il Choi1 and Jin-Baek Kim1* Abstract Background: Jacalin-related lectins in plants are important in defense signaling and regulate growth, development, and response to abiotic stress. We characterized the function of a rice mannose-binding jacalin-related lectin (OsJAC1) in the response to DNA damage from gamma radiation. Results: Time- and dose-dependent changes of OsJAC1 expression in rice were detected in response to gamma radiation. To identify OsJAC1 function, OsJAC1-overexpressing transgenic Arabidopsis plants were generated. Interestingly, OsJAC1 overexpression conferred hyper-resistance to gamma radiation in these plants. Using comparative transcriptome analysis, genes related to pathogen defense were identified among 22 differentially expressed genes in OsJAC1-overexpressing Arabidopsis lines following gamma irradiation. Furthermore, expression profiles of genes associated with the plant response to DNA damage were determined in these transgenic lines, revealing expression changes of important DNA damage checkpoint and perception regulatory components, namely MCMs, RPA, ATM, and MRE11. Conclusions: OsJAC1 overexpression may confer hyper-resistance to gamma radiation via activation of DNA damage perception and DNA damage checkpoints in Arabidopsis, implicating OsJAC1 as a key player in DNA damage response in plants. This study is the first report of a role for mannose-binding jacalin-related lectin in DNA damage. Keywords: Jacalin-related lectin (JRL), Ionizing radiation, Transcriptome analysis, DNA damage response (DDR) Background Lectins are carbohydrate-binding proteins that play diverse roles in both plants and animals [1]. In plants, lectins interact with endogenous carbohydrates and reportedly are involved in signaling pathways [2]. Twelve subfamilies of plant lectins have been identified [3]. One subfamily, the jacalin-related lectins (JRLs), is named for the presence of a jacalin-like domain and comprises 25 identified members [4]. This large subfamily has been further divided into two subgroups, based on the members’ carbohydrate-binding properties, * Correspondence: jbkim74@kaeri.re.kr † In Jung Jung and Joon-Woo Ahn contributed equally to this work. 1 Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup-si, Jeollabuk-do 56212, Republic of Korea Full list of author information is available at the end of the article subcellular localization, and molecular structures [5]. For example, mannose-binding JRLs are located in both the nucleus and the cytosol, whereas galactose-binding JRLs are located in vascular compartments [5]. Plant JRLs are important in the response to biotic stresses, such as pathogen and insect attack [6], as well as abiotic stresses, such as salinity stress [7]. Functionally, most JRLs are related to disease resistance and signaling in response to multiples stresses [8]. Particularly, JRLs with dirigent domains have been associated with plant defenses to pathogens. OsJAC1 is a mannose-binding JRL from rice (Oryza sativa). This factor contains a dirigent domain in its N-terminal region as described by Jiang et al. [9]. Overexpression of OsJAC1 suppressed elongation of coleoptiles and internodes, consistent © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jung et al. BMC Plant Biology (2019) 19:561 with a regulatory function for OsJAC1 in growth and development [10]. Furthermore, Weidenbach et al. [11] concluded that this protein is also involved in plant defense to pathogen attack. The genomes of all organisms are vulnerable to a variety of detrimental endogenous and exogenous factors, including replication errors, reactive oxygen species (ROS), ionizing radiation, and genotoxic chemicals. Ionizing radiation, which includes gamma radiation, is a carcinogen. Gamma irradiation directly damages a genome by introducing doublestrand breaks (DSBs) in the DNA [12]. Repair of DSBs occurs via two important pathways: non-homologous end joining and homologous recombination [13]. In addition, gamma radiation also indirectly induces DNA damage via the generation of ROS, which introduces different types of DNA lesions [14]. Cellular DNA damage response (DDR) mechanisms, including repair mechanisms, to maintain genomic integrity, are fundamentally conserved across all organisms [15, 16]. One important regulator of DDR is ataxia telangiectasia mutated (ATM) protein [17], which is a signal transducer that acts in response to DSBs. Ataxia telangiectasia and RAD3-related (ATR) protein is also involved in signaling in response to single-strand breaks and stalled replication forks [18]. DNA replication is important for transmission of genetic information to daughter cells and progeny; therefore, all organisms have mechanisms to protect the fidelity of DNA replication. For example, DNA damage can adversely affect the replication machinery and result in a stalled replication fork. DNA replication is initiated at numerous origins of replication in eukaryotes [19] via a two-step process. The first step is origin licensing, which starts with a pre-replicative complex in late mitosis or the G1 phase of the cell cycle [20]. The pre-replicative complex is composed of cell division 6 (CDC6), the originrecognition complex, the cell division cycle 10-dependent transcript 1 (Cdt1), and mini-chromosome maintenance proteins 2–7 (MCM2-MCM7). The second step, origin firing, begins with activation of the MCM2–7 complex. Component kinases, such as cycle dependent kinase (CDK) and Dbf-dependent kinase (DDK), that are specific to the S phase of the cell cycle are required for this origin firing step [20, 21]. In our preliminary microarray studies, differential expression of OsJAC1 was found in response to ionizing radiation (unpublished data). Several studies reported that plant JRLs are involved in responses to abiotic and biotic stress [6–8]; however, no evidence for a role of JRLs in DDR has been published. Therefore, we examined the molecular function of OsJAC1 in DDR. We sought to establish the effect of ionizing radiation and abiotic stresses on the expression of OsJAC1. We also generated transgenic OsJAC1overexpressing Arabidopsis lines that were resistant to gamma irradiation. We probed the molecular mechanism underlying OsJAC1 function on DDR using comparative transcriptome analysis of the OsJAC1-overexpressing lines. Page 2 of 16 Results Expression analysis of OsJAC1 in rice plants in response to ionizing radiation, abiotic stresses, and plant hormones We measured OsJAC1 expression over time in 2-week-old seedlings after exposure to different dosages of gamma radiation. OsJAC1 expression was greatly reduced in rice seedlings immediately after exposure at all levels of irradiation tested (Fig. 1a). Compared to untreated controls, the numbers of OsJAC1 transcripts were reduced approximately 150and 50-fold in plants exposed to 100 and 300 Gy gamma irradiation, respectively. The transcript levels were slightly increased 6, 12, and 24 h after irradiation compared to the 0-h time point (Fig. 1b-d); however, by 48 h after irradiation, we observed a greater than 2-fold induction of OsJAC1 expression in seedlings compared to levels in a non-irradiated control (Fig. 1e). Furthermore, the numbers of transcripts were increased at all doses of irradiation at 168 h (corresponding to 7 d) compared to the unirradiated control. These increases were approximately 30-, 4-, and 8-fold at 100, 200, and 300 Gy of gamma irradiation, respectively (Fig. 1f). To confirm this late induction of OsJAC1 transcript expression in response to ionizing radiation, dry rice seeds were irradiated with gamma radiation or an ion beam, subsequently germinated on MS media, and irradiated after 2 weeks. These seedlings exhibited increased OsJAC1 transcripts in response to both types of radiation (Fig. 1g, h). Additionally, OsJAC1 expression was altered by exposure to other stressors. OsJAC1 expression was also upregulated in response to salinity stress (Fig. 2a). In seedlings treated with NaCl for 6 h, we observed an approximately 8-fold increase in the number of OsJAC1 transcripts compared to untreated seedlings. The OsJAC1 transcript expression was also slightly increased after 3 h of exposure to heat stress, although no significant difference was observed after 6 or 12 h of exposure (Fig. 2b). Expression levels of OsJAC1 were also upregulated by jasmonic acid (JA) and salicylic acid (SA) treatment (Fig. 2c, d). OsJAC1 expression was approximately 40-fold higher 12 h after JA treatment, while SA treatment resulted in a 5-fold induction of OsJAC1 expression at this time point compared with levels in the untreated control. Generation of Arabidopsis OsJAC1-overexpressing lines We next sought to probe the molecular function of OsJAC1 by generating OsJAC1-overexpressing Arabidopsis lines. A schematic diagram (Fig. 3a) shows the structure of the OsJAC1-overexpressing construct in which OsJAC1 is regulated by the 35S promoter and terminator. Two transgenic lines, #16–6 and #18–2, displayed significant overexpression, approximately 70- and 130-fold, respectively (Fig. 3b). OsJAC1 overexpression was accompanied by higher levels of OsJAC1 protein in both transgenic lines than in a wild-type control (Fig. 3c). Figure 3d displays the morphology of the transgenic lines in the early vegetative growth stage, revealing Jung et al. BMC Plant Biology (2019) 19:561 Fig. 1 (See legend on next page.) Page 3 of 16 Jung et al. BMC Plant Biology (2019) 19:561 Page 4 of 16 (See figure on previous page.) Fig. 1 Expression of OsJAC1 in rice seedlings irradiated with ionizing radiation as determined with quantitative RT-PCR. a-f: Time courses of expression of OsJAC1 in 2-week-old rice seedlings after exposure to the indicated levels of gamma radiation. g, h: Expression of OsJAC1 in 2week-old seedlings from rice seeds that had been irradiated with gamma radiation (g) or with an ion beam (h) and then germinated on MS media. Values represent means ± SD (n = 3). Statistical analysis was carried out by one-way ANOVA (*p < 0.01) no obvious morphological differences in the transgenic lines in comparison to a wild-type control in the absence of exposure to radiation. OsJAC1 overexpression leads to hyper-resistance to gamma radiation We then assessed the effect of OsJAC1 overexpression on growth and development in response to gamma radiation. Transgenic lines and wild-type control plants were irradiated with 200 or 300 Gy gamma radiation, and growth rates were compared 2 weeks later. There were no morphological differences between the transgenic and control plants in the reproductive stage in the absence of irradiation (Fig. 4a). Following irradiation, the OsJAC1overexpressing lines grew faster than wild-type plants at both doses of irradiation (Fig. 4a). Consequently, the overexpressing lines were taller and accumulated more mass than the irradiated control plants (Fig. 4b, c). Specifically, both OsJAC1-overexpressing lines displayed plant heights and fresh weights that were more than 3-fold higher than those in controls after treatment with 300 Gy gamma radiation. We also measured the growth rates of OsJAC1overexpressing lines treated with NaCl as a means to impose salinity stress. OsJAC1 overexpression enhanced root growth in the stressed plants compared to unstressed plants (Additional file 1: Figure S1). Therefore, we conclude that plants with OsJAC1 overexpression possess resistances to both gamma radiation and salinity stress. Transcriptomic analysis of the DNA damage response in OsJAC1-overexpressing lines Our next step was to probe the molecular function of OsJAC1 in DDR. We performed transcriptome analysis of OsJAC1-overexpressing lines. A total of more than 129 million trimmed reads were generated from a wild-type control and two OsJAC1-overexpressing transgenic lines treated with or without gamma irradiation (Table 1). Trimmed reads were mapped to the reference gene set from the ARAPORT database (https://www.araport.org/). The average mapped rate of six samples was 84% (Table 1). Figure 5 shows the number of upregulated and downregulated DEGs in both OsJAC1-overexpressing lines compared to the wild-type Fig. 2 Time course of expression of OsJAC1 in 2-week-old rice seedlings exposed to abiotic stresses (a) salinity stress or (b) heat stress or to plant hormones (c) SA or (d) JA as determined by quantitative RT-PCR. Data represent means ± SD (n = 3). One-way ANOVA was used for statistical analysis (**p < 0.01, 0.01 < *p < 0.05) Jung et al. BMC Plant Biology (2019) 19:561 Fig. 3 (See legend on next page.) Page 5 of 16 Jung et al. BMC Plant Biology (2019) 19:561 Page 6 of 16 (See figure on previous page.) Fig. 3 Generation of OsJAC1-overexpressing Arabidopsis lines and confirmation of enhanced expression. a Schematic diagram of vector construct for OsJAC1 overexpression. b OsJAC1 transcripts in OsJAC1-overexpressing lines were detected using quantitative RT-PCR. Data represent means ± SD (n = 3). Statistical analysis was carried out by one-way ANOVA (*p < 0.01). c Expression levels of OsJAC1 in OsJAC1-overexpressing lines as determined using western blot. d Photographs of OsJAC1-overexpressing lines and wild-type plants 30 d after sowing. Note that morphologies are similar control after 100 Gy gamma irradiation. The two transgenic lines shared 12 upregulated and 10 downregulated DEGs. In upregulated DEGs, three xyloglucan endotransglucosylase/ hydrolase genes (AT4G14130, AT3G23730, and AT5G65730) were detected (Table 2). Interestingly, pathogen defenserelated genes, such as disease resistance proteins (AT5G41740 and AT5G41750) and NPR1-like protein (AT5G45110), were among the downregulated DEGs of both OsJAC1overexpressing lines. Additional file 2: Table S1 shows expression data for all annotated transcripts in OsJAC1overexpressing lines.. We next assessed the expression profile of genes involved in DNA replication in OsJAC1-overexpressing lines with and without gamma irradiation (Fig. 6). In the absence of irradiation, expression of MCM5, 6, and 7 was greater in OsJAC1-overexpressing lines than in the wild-type control. Following irradiation, the expression of MCM6 and MCM7 was significantly upregulated in OsJAC1-overexpressing lines compared to the irradiated control plant. Additionally, the transcript level of At1g23750 (replication protein A1) was significantly reduced by OsJAC1 overexpression in the absence of irradiation compared to the wild-type control. There were fewer RPA3A and RPA3B transcripts in OsJAC1-overexpressing lines without gamma irradiation compared to the wild-type control, whereas gamma irradiation resulted in transcriptional induction of these two genes (Fig. 6). Both POLGAMMA1 and the At5g67100 (DNA polymerase alpha subunit A) gene were upregulated in the transgenic lines in the absence of irradiation compared to the wild-type plants. Similarly, the expression levels of polymerase epsilon subunits TIL1 and TIL2 were increased by OsJAC1 overexpression under non-irradiated conditions, whereas slight Fig. 4 Morphological features and growth responses of OsJAC1-overexpressing Arabidopsis lines in response to gamma radiation. a Two-week-old seedlings were irradiated using gamma radiation. Photographs of OsJAC1-overexpressing lines and wild-type plants 30 d after irradiation. b, c Heights and fresh weights of OsJAC1-overexpressing lines and wild-type plants after gamma irradiation. Data represent means ± SD (n = 3). Statistical analysis was carried out by one-way ANOVA (**p < 0.01, 0.01 < *p < 0.05) Jung et al. BMC Plant Biology (2019) 19:561 Page 7 of 16 Table 1 Number of trimmed and mapped reads of wild-type and OsJAC1-overexpressing transgenic lines with/without gamma irradiation Sample Total trimmed readsa Mapped read Mapped rate (%) WT 23,191,133 19,396,927 83.6 16–5 25,199,270 21,188,297 84.0 18–2 20,500,887 18,441,540 89.9 WT (100 Gy) 19,967,350 16,002,371 80.1 16–5 (100 Gy) 19,971,641 17,370,030 86.9 18–2 (100 Gy) 21,120,649 16,840,320 79.7 Total 129,950,930 109,239,485 84.0 a All trimmed reads were summed from the two biological replicates of each sample reductions of these transcripts were observed after gamma irradiation. In addition, gamma irradiation resulted in transcriptional induction of the At1g67320 (DNA primase large subunit) gene in the transgenic lines (Fig. 6). Figure 7 displays the expression levels of genes involved in homologous recombination repair. OsJAC1 overexpression affected the accumulation of ATM. Expression of this gene was significantly upregulated in non-irradiated OsJAC1-overexpressing lines compared to the wild-type control. Interestingly, we did not detect significant differences in ATR expression between the overexpressing lines and the wild-type control (data not shown). Meiotic recombination 11 (MRE11) and Fanconi anemia group J protein were upregulated by OsJAC1 overexpression in both irradiated and non-irradiated plants (Fig. 7). Figure 8 shows the expression patterns of genes related to nucleotide excision repair, mismatch repair, and nonhomologous recombination. In nucleotide excision repair, OsJAC1 overexpression enhanced the transcriptional accumulation of DDB1A and DDB1B (UV-damaged DNA damage-binding proteins) under non-irradiated conditions (Fig. 8a). DNA mismatch repair genes MSH3, MSH6, and MLH3 were increased in both transgenic lines (Fig. 8b), and gene expression of the non-homologous recombination repair factor At4G57160 (DNA ligase 4) was increased by OsJAC1 overexpression without gamma irradiation (Fig. 8c). Discussion OsJAC1 is involved in the response to abiotic stress, including gamma irradiation and salinity stress JRLs are associated with plant responses to stress, including abiotic stresses and attack by pathogens [8]. The expression of OsJAC1, which encodes a JRL, was upregulated in a time- and dose-dependent manner following exposure to both gamma radiation and an ion beam (Fig. 1). We noted some similarities between these responses and two relevant previous studies. Jin et al. [22], using microarray analysis, observed time- and dosedependent expression of genes associated with signal transduction, transcription, and metabolism in human mesenchymal stem cells exposed to gamma radiation. These genes were either involved in cellular defense, such as apoptosis and responses to stress, or in fundamental cellular processes, such as DNA replication and repair. It has been also been noted that in Chlamydomonas reinhardtii [23], the expression of many DDR genes was altered by gamma irradiation. From the similarities between the response of OsJAC1 and these other genes to radiation, we hypothesized that OsJAC1 may participate in DDR, perhaps in signal transduction involved in these processes. Given the central role of JRLs in the response of plants to stress, we also examined the response of OsJAC1 expression to salinity stress. Salinity stress, like irradiation, increased OsJAC1 expression in rice (Fig. 2a), and OsJAC1overexpressing lines displayed resistance to salinity stress compared to a wild-type control (Additional file 1: Figure S1). Similar observations were made by Zhang et al. [7], who Fig. 5 DEG analysis of OsJAC1-overexpressing Arabidopsis lines compared to a wild-type control after 100 Gy gamma irradiation. Venn diagrams show number of upregulated (a) and downregulated (b) DEGs Jung et al. BMC Plant Biology (2019) 19:561 Page 8 of 16 Table 2 Up- and down-regulated DEGs were commonly detected in both OsJAC1-overexpressing lines Locus Up Down Fold induction Definition #16–5 #18–2 AT4G14120 2.56 2.08 Unknown AT4G14130 1.75 1.89 Xyloglucan endotransglucosylase/hydrolase 15 AT3G23730 1.57 1.54 Xyloglucan endotransglucosylase/hydrolase 16 AT2G30600 1.28 1.51 BTB/POZ domain-containing protein AT5G44130 1.22 1.19 FASCICLIN-like arabinogalactan protein 13 precursor AT2G17230 1.18 1.11 EXORDIUM like 5 AT4G25580 1.12 1.11 CAP160 protein AT3G19680 1.11 1.04 Protein of unknown function (DUF1005) AT4G16563 1.11 1.35 Eukaryotic aspartyl protease family protein AT5G46760 1.08 1.18 Basic helix-loop-helix (bHLH) DNA-binding family protein AT5G46750 1.05 1.01 ARF-GAP domain 9 AT5G65730 1.01 1.01 Xyloglucan endotransglucosylase/hydrolase 6 AT5G47910 −1.83 −1.47 Respiratory burst oxidase homologue D AT5G41750 −1.76 −1.70 Disease resistance protein (TIR-NBS-LRR class) family AT5G41740 −1.67 −1.69 Disease resistance protein (TIR-NBS-LRR class) family AT4G34150 −1.23 −1.20 Calcium-dependent lipid-binding (CaLB domain) family protein AT5G35735 −1.22 −1.46 Auxin-responsive family protein AT1G61890 −1.19 −1.50 MATE efflux family protein AT2G38470 −1.06 −1.10 WRKY DNA-binding protein 33 AT5G45110 −1.06 −1.22 NPR1-like protein 3 AT4G29780 −1.05 −1.93 Unknown AT4G33920 −0.62 −1.00 Protein phosphatase 2C family protein also identified a relationship between lectins and abiotic stresses, including salinity stress, in rice. One effect of salinity stress in plants is the generation of ROS [24], which are also generated by ionizing radiation. ROS damages cellular components, including DNA, in numerous ways [25, 26], and these similar responses further strengthen the relationship between OsJAC1 and DDR. JRLs are regulated by the plant hormones JA and SA, which are related to stress responses and pathogen defense in plants [11, 27, 28]. Thus, we examined the effect of these hormones on expression of OsJAC1. The hormones enhanced transcription of OsJAC1 (Fig. 2c, d). SA is associated with genotoxic stress that results from exposure to ethyl methanesulphonate and methyl mercuric chloride [29] and may enhance the genotoxic stress-related signaling pathway [30]; however, the role of SA in this signaling remains unclear [31]. These hormones play central roles in the plant defense response to ROS [32, 33], and their signaling pathways were affected in a dose-dependent manner by H2O2 accumulation in the cat2 Arabidopsis mutant [34, 35]. Similarly, silencing of mannose-binding lectin (CaMLB1) transcript led to a reduction in both disease resistance and ROS accumulation in pepper plants [36]. Furthermore, Weidenbach et al. [11] reported that OsJAC1 mediated the pathogen defense response in rice. Interestingly, however, DEG analysis displayed downregulation of pathogen defenserelated genes in OsJAC1-overexpressing lines (Table 2). These results suggest that OsJAC1 regulates different stresses, such as DNA damage and pathogen attack, via coordination with levels of ROS in plants. OsJAC1 overaccumulation leads to modulation of DNA replication components The relationship between OsJAC1 and abiotic stresses is well documented [7], but the molecular function of this protein has not been established. We first probed the molecular function of OsJAC1 in DDR following exposure of plants to gamma radiation. Arabidopsis lines overexpressing OsJAC1 showed tolerance to gamma radiation (Fig. 4). In addition, DEG analysis revealed that these transgenic lines highlighted differential expression of genes involved in pathogen defense after gamma irradiation (Fig. 5 and Table 2). OsJAC1 functions in pathogen defense have been well characterized previously [11]. Hadwiger et al. [37] also reported that DDR is closely associated with pathogen defense via SA signaling. Thus, differential expression of pathogen-related Jung et al. BMC Plant Biology (2019) 19:561 Page 9 of 16 Fig. 6 Comparative transcriptome expression profiles of genes involved in DNA replication from OsJAC1-overexpressing lines and a wild-type control before and after gamma irradiation Jung et al. BMC Plant Biology (2019) 19:561 Page 10 of 16 Fig. 7 Comparative transcriptome expression profiles of genes associated with homologous recombination from OsJAC1-overexpressing lines and a wild-type control with and without gamma irradiation