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Deciphering the molecular transcriptomic mechanisms of carbon ion beams and X-ray on rice seedlings

BMC Genomics volume 26, Article number: 308 (2025) Cite this article

Abstract

Background

Ionizing radiation (IR) is an abiotic stress factor that can be not only a means to explore plant resistance but also a potent mutagen in agricultural breeding. The diverse physical parameters of different types of IR result in varying effects on plants, which in turn leads to differences in the spectrum of genetic variations in the offspring. Investigating plant response mechanisms to different IR is crucial for enhancing plant resistance and comprehending of the differences in mutation generation from various physical mutagenic sources in mutation breeding. Nevertheless, the mechanism underlying the complex responses of plants to different IR are not yet fully comprehended.

Results

we conducted transcriptome sequencing on rice seedlings that exhibited a relative root length of approximately 69% after being exposed to carbon ion beams (CIBs) and X-ray respectively. The results revealed that X-ray induced a greater number of differentially expressed genes (DEGs) than CIBs, with 5681 and 2198 DEGs were identified respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that DNA replication, damage repair, phytohormone signaling, and antioxidant pathways were implicated in the response of rice seedling to IR. These pathways demonstrated diverse response patterns following different IR. Additionally, through two IR with different linear energy transfer (LET), we found some common DEGs that contribute to the radiation response in rice seedlings, such as LOC4331062, LOC4333870.

Conclusion

This study offers insights into the molecular transcriptomic mechanisms underlying the impacts of IR on rice seedlings. It provides a new perspective for further exploration of irradiation-induced damage repair factors and understanding the reasons for the differences in mutations created by different mutagenic sources in plants.

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Background

Nowadays, the perpetually evolving global climate imposes significant pressure on living organisms. Comprehending how these organisms adapt and withstand such alterations is crucial for both environmental conservation and agricultural breeding [1]. Ionizing radiation (IR) is an abiotic stress factor that can be not only a means to explore plant resistance but also a potent mutagen in agricultural breeding. IR induces reactive oxygen species, interferes with plant growth and reproduction, and cause DNA damage indirectly or directly, thereby resulting in gene mutation and even the evolution of plant genomes [2, 3].

Unlike animals, plants have adopted a sessile lifestyle to adapt diverse environments. Consequently, plants have evolved a unique response and defense mechanisms to recognize and cope with abiotic stresses like IR. Plants possess a higher radiation tolerance than animals and can endure doses that are lethal for many mammals. At same irradiation dose, plant cells generate one-third of the DNA double-strand breaks (DSBs) that animal cells produce [4]. Despite extensive research on plant growth, DNA damage repair, and gene expression after radiation, studies on plant radiation effects are still less comprehensive than those on animals [5,6,7,8]. Moreover, the mechanism of a series of complex responses to radiation in plants is not fully comprehended. Therefore, a deeper understanding of plant responses to IR is crucial for comprehending DNA repair mechanisms and how plants maintain growth after abiotic stresses like IR, providing a scientific basis for improving plant resilience.

IR is an effective method for inducing mutations in plants [9]. Although these mutations occur randomly, resulting in both positive and negative effects, they are also the important resource of the genetic diversity in plants. In mutation breeding, we conventionally screen mutants follow the specific breeding goals to rationally use these mutations, and finally improve crop varieties, resistance, and productivity. Currently, more than 800 crop varieties have been developed globally through mutation breeding, primarily using acute radiation (https://data.apps.fao.org/catalog/dataset/mutant-variety-dataset). Different types of IR have different effects on plants due to their distinct physical parameters. Linear energy transfer (LET) refers to the average energy expended per unit length of an ionized particle's trajectory. Different types of IR cause various DNA damage and physiological damage due to their different LET [10], leading to different repair pathways in organisms, resulting in correct repairs, mutation, or death [11, 12]. Studies on mammalian cells suggest that exposure to high- and low- LET radiation leads to distinct spatial distributions of key factors involved in repairing DSBs, and high-LET IR kills more cells at the same doses compared to low-LET by inhibiting only classical non-homologous end-joining (cNHEJ) [13, 14]. Previous studies have demonstrated that different LET radiations produced distinct mutation spectra in plant progeny, especially in later generations. For example, γ-ray irradiation (a type of low-LET IR) in M5 generation induced more small mutations than CIBs irradiation (a type of high-LET IR) [15]. In high-generation offspring of rice, CIBs induced an increase in Insertions and Deletions (InDels), while γ-ray induced an increase in Multi-nucleotide variants (MNVs) [16]. The process by which radiation induces mutations involves material interactions, plant response in M1 generation, and genetic variation in offspring. However, the response mechanisms of plants after exposure to different LET radiations are not completely understood.

Therefore, we selected high-LET heavy ion beams and low-LET X-ray for irradiating rice seedlings respectively. We performed transcriptomic sequencing to identify genes that exhibited distinct expression patterns and shared response factors after exposure to different types of IR. Our objective was to found the key genes in plants that potentially participate in responding to different IR induced- abiotic stress, and to provide a new perspective for understanding the mutational differences caused by different IR. Meanwhile, this research provides essential data on different qualities but also offers valuable insights for evaluating plant growth in the contexts of deep-space exploration.

Methods

Plant materials and growth conditions

Kitaake rice seeds, which were disease-free, uniformly plump, and from the same batch, were chosen for germination. The dry seeds were initially sterilized by soaking in a 3% H2O2 solution for 30 min and subsequently rinsed 3–5 times with water. The seeds were placed in a 90 mm circular petri dish containing three layers of moist filter paper and cultured for 5 days. Refer to the rice growth cultivation method of Ronald Laboratory (https://kitbase.ucdavis.edu/), during the first 1–4 days, they were germinated in an incubator at a constant temperature (32℃, no light), and then transferred to a light incubator(temperature 28–30℃, light intensity 250 μmol/m2·s, light cycle 14 h light/10 h dark) for one day. Seedlings that had germinated uniformly were selected and placed in a 35 mm circular petri dish containing moist filter paper, with five seedlings per dish, and sealed with Parafilm film.

High-energy heavy ion beams and X-ray radiation treatments

The prepared rice seedling samples were subjected to radiation treatment. The heavy ion beam was provided by the Heavy Ion Research Facility in Lanzhou (HIRFL) of the Institute of Modern Physics, Chinese Academy of Sciences. The high-energy carbon ion (12C6+) beam from the accelerator had an energy of 80.55 MeV/u, a LET of 34 keV/μm, and a dose rate of approximately 60 Gy/min. Samples were irradiated at Irradiation Terminal for Life science and Shallow-seated Tumor Therapy (TR4). The radiation doses were set at 5, 10, 15, 20, 25, 30, and 40 Gy. X-ray were used for radiation mutagenesis of the samples using the X-ray irradiator (X-RAD255) of the Public Technology Service Center of the Institute of Modern Physics, Chinese Academy of Sciences. The dose rate was about 5.5 Gy/min, with radiation doses of 10, 20, 30, 40, 50, 60, and 80 Gy.

Determination of growth and development index

The root length of the plants was measured 6 days after radiation treatment. The root length measurement is the distance from the root tip to the bottom of the seedling, calculated in centimeters. For each treatment group, 20–30 seedlings were selected for digital photography, and this process was repeated three times. The length was measured using ImageJ software. The experimental data are expressed as mean ± standard deviation. For the analysis of significant differences, SPSS statistical software was used to perform a one-way ANOVA to analyze statistical differences, followed by Duncan's test (p < 0.05*). Graphs were created using Origin software (version 8.5) and Excel 2016, and images were combined using Photoshop 2024.

RNA extraction, library preparation, RNA sequencing

For extracting RNA sample, Kitaake rice seedlings treated with 20 Gy carbon ion beam and 40 Gy X-ray radiation at 2, 6, and 24 h post-irradiation was used as the transcriptome sequencing materials, with the non-irradiated group as a control. A mixture of shoots from 5–10 rice seedlings was sampled, with each sample set up in triplicate for biological replication. The samples were immediately placed in liquid nitrogen, then transferred to a −80℃ freezer for storage.

Total RNA was extracted using Trizol reagent (TaKaRa, Inc., Dalian, China). DNase I treatment was performed to eliminate DNA contamination in the RNA samples. The RNA quality was analyzed using Agilent 2100 and NanoDrop (all sample’s RIN ≥ 9.1). mRNA was enriched with Oligo (dT) magnetic beads and fragmented into short pieces. Second-strand cDNA was synthesized via reverse transcription with random hexamer primers. After end repair, the 5' tail was phosphorylated, and adenine was added to the 3' tail. Sequencing adapters were ligated to the double-stranded DNA fragments. We used 1 μg of RNA for library construction for each sample. The cDNA fragments were amplified by PCR to construct the cDNA library. The construction of the cDNA library, quality control, and Illumina sequencing of rice seedling samples were outsourced to Beijing Genomics institution (BGI), using paired-end sequencing. Transcriptome data were uploaded to the Genome Sequence Archive (GSA) database of the National Center for Biotechnology Information, under accession number CRA014790.

Bioinformatic analysis

The raw sequences obtained from sequencing require processing to filter out reads containing adapters, unknown bases (with N content greater than 10%), and low-quality bases by SOAPnuke(v1.4.0). The filtered sequences of higher quality are termed clean reads and are saved in FASTQ format. The mean of Q20 and Q30 for all sample’s clean reads were higher than 90%. The high-quality data post-filtering was used for subsequent analysis. The clean data were mapped to the reference genome by HISAT (v2.1.0). In this experiment, the rice genome (Oryza sativa Japonica Group) in the NCBI database was used for sequence alignment and subsequent analysis (https://www.ncbi.nlm.nih.gov/assembly/GCF_001433935.1).

The expression levels of transcripts or genes are quantitatively assessed using Fragments per Kilobase of Exon per Million Fragments Mapped (FPKM). We divided the 24 transcriptome samples into eight groups, each with three biological replicates, based on the type of radiation and sampling time post-irradiation. These groups included rice seedlings not irradiated and those sampled at 2, 6, and 24 h after exposure to CIBs and X-ray. Both CIBs and X-ray treatments have their respective non-irradiated control groups for comparison. After exposure to the two types of IR, these samples were then compared with their corresponding control group to screen for DEGs Differential expression analysis was using the phyper function in R language to calculate P value, followed by correction of the P value with the Q value software package. The Log2 (Fold Change) was set as ≥ 1 and the significance Q value was set as ≤ 0.05. DEGs were screened based on the negative binomial distribution principle using DEseq2 software(R4.1.2) [17]. Significantly enriched GO terms were identified using the GO (Gene Ontology) database (http://www.geneontology.org/), thereby providing biological functions significantly associated with candidate genes. Enrichment analysis of DEGs was conducted based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.genome.jp/), and annotation results were obtained using KOBAS2.0 software [18]. Pathway enrichment analysis using the R package clusterProfiler 4.0. [19]. We selected the top twenty enriched pathways for each ray and each time period for display. Among the pathways presented, the Q-values were all less than or equal to 0.15. The FPKM trend of genes in rice seedlings at three time points was analyzed on the Omicshare Tools website (https://www.omicshare.com/).

Quantitative real-time PCR

The cDNA template for RT-qPCR was synthesized from RNA samples using the reverse transcription kit iScriptTM cDNA synthesis kit (Bio-RAD), in accordance with the instructions provided. Eleven differentially expressed genes were randomly selected to design gene fluorescence expression primer sequences using the Oligo Architect online program (http://www.oligoarchitect.com/LoginServlet). The list of primers can be found in Table S6. The SsoFastTM EvaGreen® Supermix (Bio-RAD) fluorescent quantitative reagent kit was used for detection. Actin was used as an internal reference gene, and the quantitative transcription level of each gene was normalized using the 2−ΔΔCTmethod to calculate the relative expression level of the gene.

Results

IR affects the growth of root length in rice seedlings

The growth status of seedlings following radiation reveals the extent of radiation impact on plant growth. We discovered that the root length of rice seedlings exhibited a decreasing tendency with the escalation of doses of two different types of IR (Fig. 1A, and B). The inhibitory effects of 20 Gy CIBs and 40 Gy X-ray on rice root growth were comparable, accounting for 69.54% and 68.53% of the control group, respectively, and both differed significantly from the control (Fig. 1C). Our study suggests that the RBE of CIBs radiation on rice is twice that of X-ray. To explore the response of different types of radiation on the gene expression level of rice, we selected these two doses for transcriptome analysis.

Fig. 1
figure 1

Relative root length of rice seedlings after CIBs and X-ray irradiation. A The relative root length of rice seedlings 6 days after CIBs; B The relative root length of rice seedlings 6 days after X-ray

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Overview of gene expression in rice seedlings after different IR

We conducted the RNA-seq of rice seedling collected 2, 6, 24, and 72 h after 20 Gy CIBs and 40 Gy X-ray radiation. This dataset consisted of 24 samples with 44.33 million clean reads, and each sample contained 6.65 G of clean bases with Q30 quality scores of ≥ 90.67% (Table S1). The majority of clean reads were mapped to the rice genome, including 89.23%−91.4% unique mapped reads. Correlation analyses showed that the three sets of biologically replicated samples were highly reproducible and consistent (Fig.S1). We also conducted a PCA analysis, and the results indicated good repeatability within groups and clear discrimination between groups (Fig.S2). To validate the accuracy and reliability of the transcriptome data, we performed RT-qPCR analysis and compared the RNA-seq data with the RT-qPCR results. The RT-qPCR results were highly correlated with the RNA-seq results (R2 = 0.9266) (Fig.S3). These findings indicated that the transcriptome results possess high reliability.

Based on the screening criteria of FC ≥ 2 and Q value ≤ 0.05, differential expression gene analysis revealed that rice seedlings generate more DEGs after X-ray than after CIBs irradiation (Fig. 2A, and B, Fig.S4). The number of DEGs at 2, 6 and 24 h after X-ray radiation was 45.5%, 75.9% and 69.4% higher than that of CIBs, respectively. This result suggested that the impact of X-ray on the transcription level of rice seedlings was greater than that of CIBs with the same biological effects. In both irradiated rice seedling, the peak number of DEGs was observed at 2 h. However, the number of DEGs at the 6 h for CIBs was lower than that at 24 h, while the trend was reversed for X-ray (Fig. 2A, and B, Fig.S4). The sequence of the number of up-regulated DEG at three times for both radiation treatments was 2 h > 24 h > 6 h. For CIBs, the number of down-regulated DEGs decreased with time. In contrast, for the X-ray, the peak number of down-regulated DEGs occurred at 6 h. Venn diagrams (Fig. 2C, and D) shows that after CIBs radiation, 94 genes were identified as DEGs at all three time points, with 932 unique DEGs at 2 h, more than at 6 h (138) and 24 h (553). For X-ray radiation, 1573 unique DEGs identified at 24 h, more than at 2 h (1292) and 6 h (1030) post-radiation. In summary, the gene expression levels in rice seedlings responded differently at various times after IR, and X-ray radiation induced more substantial transcriptional changes than CIBs.

Fig. 2
figure 2

Gene expression differences in rice seedlings after CIBs and X-ray irradiation. A The number of differentially expressed genes in rice seedlings at 2 h, 6 h, and 24 h after CIBs; B The number of differentially expressed genes in rice seedlings at 2 h, 6 h, and 24 h after X-ray radiation; C Venn diagram of differentially expressed genes in rice seedlings at 2 h, 6 h, and 24 h after high-energy CIBs radiation; D Venn diagram of differentially expressed genes in rice seedlings at 2 h, 6 h, and 24 h after X-ray radiation

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GO and KEGG analysis after different IR

To determine the function of the identified DEGs between the control and CIBs irradiation treatment samples of rice seedling, we performed GO and KEGG enrichment analysis. The GO classification results showed that the main entries of biological process (BP) ontology for both radiation-induced DEGs were cellular processes, metabolic processes and biological processes. The main entries of cellular component (CC) ontology were cell part, organelle, cell membrane. Binding, transcription regulator activity, and molecular function regulation were the main entries in molecular function (MF) ontology (Fig.S5, Table S2). A smaller percentage of DEGs were annotated to cellular processes and metabolic processes after CIBs irradiation of rice seedlings compared to X-ray. GO enrichment analysis indicated that GO entries significantly enriched after both CIBs and X-ray radiation at 2 h include nuclear assembly, DNA replication, and cell cycle in the biological process; at 6 h, they were mainly related to nuclear assembly and cell cycle; at 24 h, they were primarily associated with "photosynthesis" (Fig. 3A, Fig.S6). In cellular components, at 2 h they include nucleosomes; at 6 h, they are mainly related to photosynthetic processes such as chloroplasts; at 24 h, they were primarily associated with chloroplasts and cytoplasm (Fig. 3A). In molecular functions, at 2 h they included "ATP binding", "calmodulin binding", "DNA binding", "protein heterodimerization activity", "protein kinase activity", "protein serine/threonine kinase activity", and "sequence-specific DNA binding"; at 6 h they included "microtubule binding" and "microtubule motor activity"; at 24 h "oxidoreductase activity". In summary, GO enrichment analysis revealed that CIBs mainly affect biological processes such as microtubules and photosystems, whereas the X-ray treatment group has greater influences on biological processes like ribosomes and cytoplasm.

Fig. 3
figure 3

Enrichment analysis of differential genes in rice seedlings CIBs and X-ray irradiation. A GO enrichment of differential genes in rice seedlings after different IR. B KEGG enrichment of differential genes in rice seedlings after different IRs. The X-axis represents Rich ratio, represents the number of differentially expressed genes annotated in this pathway divided by all genes identified in that pathway. The higher the value, the larger the proportion of differentially expressed genes annotated for that pathway. The size of the circle indicates the number of differentially expressed genes annotated for that pathway

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The KEGG enrichment analysis results indicated that after CIBs and X-ray radiation, pathways such as alpha-linolenic acid metabolism, DNA replication, glutathione metabolism, MAPK signaling, meiosis, plant hormone signal transduction, and plant-pathogen interaction pathways significantly enriched at 2 h (Fig. 3B). At 6 h, the most significantly enriched pathways were DNA replication, glutathione metabolism, photosynthesis, and photosynthesis—antenna proteins. However, at 24 h after X-ray irradiation, the most significantly enriched pathway was ribosome, while after CIBs irradiation, the most significantly enriched pathway was diterpenoid biosynthesis. In summary, both types of radiation jointly influence biological processes such as plant hormone signal transduction, DNA replication, oxidoreductase activity, and photosynthesis in rice seedlings. However, the DEGs induced by CIBs were more inclined to be enriched in pathways like carbon metabolism and microtubules, whereas X-ray is more prone to be enriched in ribosomes pathway.

Overlapping transcriptional responses among different IR

We analyzed gene expression trends over time after different IR, and these DEGs were divided into 20 clusters with different dynamic expression patterns (Fig. 4A). Among them, six clusters were significant (cluster3, 6, 10, 14, 17, 19), corresponding to gene expression down-regulated at 2 h, up-regulated at 24 h, down-regulated at 2 h and 6 h, up-regulated at 24 h, only up-regulated at 24 h, up-regulated at 2 h-down-regulated at 6 h-unchanged at 24 h, only up-regulated at 2 h, and continuous up-regulation of gene expression. Cluster14 was the most enriched following both CIBs and X-ray exposure, indicating an initial biological response in plants to IR. Cluster19 also played a crucial role in the post-radiation response as its gene expression level increased over time. Meanwhile, the DEGs detected in these two clusters were further subjected to KEGG pathway enrichment analysis (Fig. 4B; Fig.S7). The results showed that the DEGs in cluster14 were mainly associated with plant-pathogen interaction and MAPK signaling pathway-plant and plant hormone signaling pathways, while those in cluster19 were associated with Plant hormone signal transduction, MAPK signaling pathway, fatty acid elongation, glycerolipid metabolism, and glutathione metabolism pathways.

Fig. 4
figure 4

Expression tendency and pathway enrichment analysis of rice seedling CIBs and X-ray irradiation. A Cluster analysis of DEGs displaying a log2-fold change (with absolute value > 2) of transcripts after IR stress in 2 h、6 h and 24 h. B Cluster 14’s bubble diagram of significant enrichment pathways after IR. The colour of the circles represents Q value, and the size of the circles indicates the number of differentially expressed genes annotated for that pathway

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Expression profile of DNA replication and damage repair genes after different IR

IR causes direct or indirect DNA damage in plants. Consequently, we concentrated on the expression profiles of genes related to DNA replication and repair in rice seedlings after different radiation at three time points. After two types of IR, the expression of many genes significantly changed significantly at 2 h and 6 h. However, only 3 up-regulated genes were identified in the CIBs group and only 4 up-regulated genes in the X-ray group at 24 h (Fig. 5, Table S3). This suggested that the genes associated with DNA replication and repair are most active at 2 and 6 h after radiation, indicating that the repair time of DNA damage after acute irradiation was mainly within one day.

Fig. 5
figure 5

Response patterns of DNA replication/repair genes to CIBs and X-ray irradiation. The fold change in gene expression was represented by color intensity, and blue represents down-regulated genes. “*” means the gene is significantly different, (Log2 (Fold Change) ≥ 1, Q value ≤ 0.05)

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DNA damage repair mainly comprises five types: base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end joining (NHEJ). In BER, uracil-DNA glycosylase and the mitochondrial gene were down-regulated by both irradiations at 2 h, and X-ray induced a significant downregulation of an adenosine glycosylation enzyme. At 6 h, the above glycosylation enzymes were down-regulated by both CIBs and X-ray; Poly-(ADP-ribose) polymerase (PARP) genes were significantly up-regulated at all three time points. In NER, most of the genes were down-regulated by both treatments at 2 h and 6 h. In MMR, both irradiations led to the down-regulation of the MSH6 and MSH2 genes at 2 h and 6 h. Notably, the proliferating cell nuclear antigen (PCNA) gene belonging to the DNA polymerase epsilon complex was significantly up-regulated only at 2 h. HR is an essential pathway for DSBs repair after radiation. In HR, CIBs induced significant down-regulation of the DNA polymerase delta small subunit-like gene, and two DNA repair and recombination protein RAD54-like genes belonging to the Rad54 family proteins were significantly up-regulated at 2 h after both IR treatments, while the gene for BRCA1-related RING structural domain protein 1 belonging to the BRCA1 core complex was significantly down-regulated. The BRCA1 core complex gene was significantly up-regulated after CIBs at 6 h, and the genes for DNA repair protein RAD51 homolog A, DNA repair protein recA homolog3, and BRCA1-associated RING domain protein 1 were significantly down-regulated after x-ray treatment, whereas LOC4332589 genes belonging to the BRCA1-B complex were significantly up-regulated. Interestingly, both radiations resulted in significant up-regulation of genes belonging to the HR pathway protein Chromatin remodeling 35 and DNA repair protein RAD51 homolog B at all three time points.

The results implied that rice seedlings will repair DNA damage within 1 day after exposure to IR. Additionally, the gene expression profiles of DNA damage repair pathways were similar, but X-ray irradiation caused higher expression changes of genes related to DNA damage repair in plants.

Plant hormone signal transduction is involved in rice seedlings’ response to IR

Plant hormones facilitate plants' adaptation to adverse environments, making the complex hormone signal pathway an ideal mediator of defense responses [20]. Therefore, we summarized the expression of 75 DEGs related to plant hormone signaling transduction in rice seedlings after radiation (Fig. 6, Table S4). Notably, the number of up-regulated and down-regulated genes in rice seedlings irradiated with X-ray were higher than those irradiated with CIBs. The number of genes associated with plant hormone signaling transduction pathways at 6 h post-irradiation was fewer than those at 2 h and 24 h. At 6 h after CIBs, only LOC4342318 showed significant upregulation, while LOC4336439 and LOC4340708 showed significant downregulation. Based on enrichment analysis of gene expression trends, we specifically focused on the immediate expression of plant hormone signaling transduction: the results revealed a significant upregulation of jasmonate ZIM-domain (JAZ) genes at 2 h after both types of IR.

Fig. 6
figure 6

The gene expression pattern of hormone signals in rice seedling CIBs and X-ray irradiation. A–H are auxin, cytokinine, gibberellin, abscisic acid, ethylene, brassinosteroid, jasmonic acid, and salicylic acid signal transduction pathways, respectively. Blue boxes represent DEGs. The fold change in gene expression was represented by color intensity. Red and blue represent upregulation and downregulation in the heatmap, respectively; “*” means the gene is significantly different, (Log2 (Fold Change) ≥ 1, Q value ≤ 0.05)

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Regarding auxin-responsive genes, in two types of radiation-treated rice seedlings, the expression of two auxin influx carrier AUX1 genes was significantly down-regulated at 2 h; the Auxin/Indole-3-Acetic Acid (Aux/IAA) gene and Gretchen Hagen3 (GH3) gene were significantly regulated after X-ray. Two small auxin up-regulated RNAs (SAUR) were up-regulated at 2 h after X-ray, and three SAURs were up-regulated at 2 h after CIBs. In the signal transduction of cytokinin, the downstream response regulator A-ARRs were down-regulated at 2 h and 6 h, and began to be up-regulated at 24 h. Abscisic acid (ABA) is a key hormone for plants to address abiotic stress. Under abiotic stress, plants exhibit a rapid accumulation of ABA, which in turn activates their stress resistance responses. The PYR/PYL in the ABA signaling pathway was down-regulated, and the protein phosphatase 2C (PP2C) was generally up-regulated. Two SnRK2 genes were significantly up-regulated at 6 h after X-ray, indicating that ABA plays a positive role in plant responses to radiation. The ethylene-responsive ethylene receptors (ETR) gene was significantly down-regulated at 2 h after X-ray; the CTR gene was significantly up-regulated at 24 h after X-ray treatment. The Ethylene insensitive 3 (EIN3) Ethylene and Ethylene insensitive 3 binding F-box (EBF1/2) were significantly regulated at 2 h after X-ray, and Ethylene responsive factor (ERF1/2) was significantly up-regulated at 2 h after both CIBs and X-ray. These findings indicated that ethylene signaling plays a crucial role within the initial 2 h after IR exposure, particularly during the early stages after X-ray. We also found that the cyclin D3 (CYCD3) gene was down-regulated at 2 h after CIBs and significantly down-regulated at 2 and 6 h after X-ray treatment. Jasmonic acid is an important mediator in plant defense against abiotic stresses. This study showed that JAZ in the jasmonic acid pathway was up-regulated at 2 h after both CIBs and X-ray, and significantly down-regulated at 24 h after X-ray, suggesting that the jasmonic acid pathway is also involved in the early stages of radiation response. The NPR1 gene was significantly up-regulated at 2 h after both types of radiation treatments, PR-1 gene was up-regulated after both types of radiation treatments, especially significantly up-regulated after 24 h of X-ray treatment, and TGA related genes were basically up-regulated after X-ray treatment, but interestingly, LOC4340708 was down-regulated in all treatment groups. We found that this gene is related to defense response. These results suggested that rice seedlings coordinate their responses to radiation damage through multiple plant hormones.

Radiation changed the gene expression of the antioxidant response in rice seedling

To understand the regulation of antioxidant activity elicited by CIBs and X-ray radiation, we analyzed the expression patterns of DEGs related to antioxidant activity (Fig. 7, Table S5). The findings revealed that the gene expression patterns differed following two types of IR. In rice seedlings after X-ray irradiation, at 2 h, 6 h, or 24 h post-irradiation, more genes related to antioxidant activity were significantly regulated compared to those after CIBs. Specifically, there were 9 up-regulated and down-regulated genes respectively at 2 h; 14 up-regulated genes and 13 down-regulated gene at 6 h; 22 up-regulated genes and 9 down-regulated genes at 24 h after X-ray radiation. In contrast, after CIBs radiation, there were 5 up-regulated genes and 3 down-regulated genes at 2 h; 5 up-regulated genes and 4 down-regulated genes at 6 h; 3 up-regulated genes and 3 down-regulated genes at 24 h. Consequently, X-ray induced more genes related to antioxidant enzyme activity.

Fig. 7
figure 7

Response patterns of antioxidant response genes to CIBs and X-ray irradiation The fold change in gene expression was represented by color intensity, red represents up-regulated genes, and blue represents down-regulated genes. “*” means the gene is significantly different, (Log2 (Fold Change) ≥ 1, Q value ≤ 0.05)

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The DEGs involved in the regulation of antioxidant activity exhibited a specific preference for different radiation. For instance, the genes associated with ascorbate peroxidase, L-ascorbate peroxidase 2, cytosolic-like, were down-regulated after CIBs but notably up-regulated after X-ray. After X-ray irradiation, a great number of genes involved in glutathione metabolism were up-regulated at both 6 h and 24 h (APX8, LOC4333870, APX2). Conversely, after CIBs radiation, the putative respiratory burst oxidase homologs H was significantly down-regulated in rice seedling at 2 h and 6 h. This suggested that X-ray exposure mainly affects the expression of antioxidant enzymes such as antioxidant enzyme (APX). After different IR treatments, the expression of many peroxidase (POX) related genes in rice seedlings showed similar trends.

Discussion

Plants have evolved multiple strategies to cope with abiotic stress [21, 22]. The exposure of a biological system to IR activates several processes between the initial energy absorption and the eventual biological damage: In direct action, radiation energy is deposited directly in the target; in indirect action, IR excites and ionizes water, producing free radicals •OH and H•, which eventually produce reactive oxygen species (ROS) [23,24,25]. Plants have a very complex and orderly mechanism to deal with IR. Hence, three time points were selected in this study to explore the staged alterations in gene expression levels in plants upon IR exposure. In this study, by analyzing the expression patterns of DEGs (Fig. 4A), we found that the highest expression levels at 2 h post-IR were mainly enriched in plant-pathogen interaction, MAPK signaling pathway-plant, and plant hormone signal transduction. Our results indicate that radiation induces a series of responses in plants: IR causes DNA damage, triggers DNA repair responses, and activates signal transduction processes. Ultimately, due to ROS, antioxidant enzyme systems are activated, further eliciting stress responses related to MAPK and hormone pathways (Fig. 8).

Fig. 8
figure 8

Prospective plant response after IR in rice seedling. The red font means up-regulate, blue font means down-regulate, orange font means both. The light orange border represents the DNA damage repair; light blue border represents plant hormone signaling; purple border represents the oxidoreductase activity; grey block means the enzyme which participate in this process

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IR can cause diverse forms of DNA damage, including DSBs, single-strand breaks (SSBs), base damage, and DNA–protein cross-linking [26,27,28]. SSBs repair pathways encompass BER, NER, and MMR, whereas DSBs are primarily repaired by HR and NHEJ [29]. In this study, DEGs after CIBs and X-ray were significantly enriched in pathways associated with nucleosome assembly, DNA replication, and cell cycle at 2 h (Fig. 3). The DNA replication licensing factor MCM gene was significantly down-regulated at both 2 h and 6 h after IR, suggesting that the cell cycle was impacted. This finding is line with prior reports on the effects of CIBs on Taraxacum kok-saghyz Rodin (TKS), indicating that plants might encounter obstructions in DNA replication and cell cycle processes due to DNA damage in the early stages (2 h and 6 h) following radiation [30]. Consequently, we concentrated on genes related to DNA damage repair pathways and discovered that the DNA repair program responded highly actively within a short period after radiation. Previous studies have demonstrated that Proliferating Cell Nuclear Antigen (PCNA) is an evolutionarily conserved protein that is associated with numerous crucial cellular processes, including DNA replication, chromatin remodeling, DNA repair, sister chromatid cohesion, and cell cycle [31, 32]. At 2 h after radiation, the PCNA gene (LOC4331062) was significantly up-regulated under both treatments, which is consistent with previous reports in Gamma rays and Platycodon grandiflorus [33, 34]. Studies have indicated that DSBs induced by high LET heavy ion beams irradiation are preferentially repaired by HR, a non-precision modality, compared to X-ray or Gamma ray irradiation [35]. Genes associated with HR at 2 h were significantly up-regulated (LOC4330819, LOC4332589, LOC4339293, LOC4352257, LOC9267584), indicating that numerous DSBs were generated after CIBs radiation and X-ray radiation, and HR-related genes play a significant role in repairing DSBs induced by both types of radiation. In this study, PARP2-A (LOC4326479) was up-regulated at 3 time points in rice seedling after IR. The PARP-2 gene has been confirmed to play a role in NHEJ induced after DSBs in both mammals and plants [36, 37]. In summary, after exposure to radiation, plants counteract various types of DNA damage caused by radiation through the synergistic effect of multiple DNA repair pathways.

Phytohormones play a crucial role in diverse physiological, biochemical, and stress responses of plants, while IR can bring about substantial alterations in the synthesis, metabolism, and signal transduction of phytohormones in plants [38, 39]. In this study, the main hormone signaling pathways in response to IR were cytokinin (CTK), auxin (Indole-3-acetic acid, IAA), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene (ETH) acid (SA), salicylic acid (SA) and ethylene (ETH). The expression trends of DEGs indicated that the significantly up-regulated DEGs at 2 h after both radiation treatments were enriched in the plant hormone signaling pathway. Therefore, we concentrated our study on the expression of plant hormones at a 2 h interval after IR exposure. Previous studies have suggested that JA has the function of inducing the expression of plant defense genes to coordinate stress and growth responses [40]. Our findings revealed that the expression of JAZ (LOC4348533) in the jasmonic acid (JA)-mediated signaling pathway was notably up-regulated at 2 h and down-regulated after that. JAZ acts as jasmonate (JA) co-receptor and transcriptional repressor in JA signaling in plants [41]. Thus, we speculate that the jasmonic acid (JA)-mediated signaling pathway exerts a negative regulatory role at 2 h, and then with the downregulation, possibly degradation, of JAZ expression, it indicates that the jasmonic acid (JA)-mediated signaling pathway begins to participate in stress signal transduction. NPR1 is crucial for transducing the SA signal, and its overexpression suppresses rice growth and development by disrupting the auxin pathway [42, 43]. In our study, the NPR1 gene (LOC4327315) and the GH3.8 gene (LOC4343785) were significantly up-regulated at 2 h after exposure to both radiation types. This suggests that the SA pathway, involving NPR1, enhances the defensive response of rice seedlings under abiotic stress, thereby weakening or even inhibiting growth. ABA plays a diverse range of roles in plant growth and development, with its primary function being to regulate plant water balance and osmotic stress tolerance [44]. Protein phosphatases of type 2C (PP2Cs) are negative regulators in the ABA signaling pathway. ABA binds to its receptor proteins RCAR/PYR/PYLs to inhibit the phosphorylation of PP2Cs protein, facilitating the transcription of ABA response genes [45]. The gene expression of PP2Cs was significantly up-regulated at 2 h after X-ray radiation and 24 h after CIBs radiation, indicating that ABA signaling played regulatory role at different stage of IR exposure. In summary, after suffering from IR exposure, plants will promptly adjust plant hormone signals to regulate plant status, and maintain internal balance, and this process will persist for an extended time.

One of the indirect effects of IR on plants is the generation of primary and secondary ROS (•OH, H•, H2O2, O2•, HO2•) that pose a threat to plant growth and reproduction [46, 47]. ROS influence the expression of a significant number of genes involved in various aspects of growth and stress responses. The plant initiates an antioxidant enzyme defense system to counteract the toxicity of excessive ROS [48, 49], playing a crucial role in plant’s resistance to radiation and subsequent growth. It has been reported that an increased activity of antioxidant enzyme is vital for facilitating the growth of Arabidopsis seedlings under low-dose CIBs radiation [50]. SOD catalyzes the conversion of superoxide anions into hydrogen peroxide and oxygen, while CAT and POD decompose hydrogen peroxide [51, 52]. Our results indicate that the most significant changes occurred in POD, followed by CAT and SOD. This phenomenon might be due to radiation-induced hydrogen peroxide production triggering a strong response. Our study discovered that some up-regulated DEGs in rice seedlings after IR were located in pathways related to antioxidant metabolites, such as glutathione metabolism. With the prolongation of time rice seedlings exhibit distinct antioxidant response patterns after two types of IR: The X-ray group has an increased number of genes associated with antioxidant activity and a relatively strong response, while the CIBs group has a decreased number of genes and a relatively weak response. Ascorbate Peroxidase (APX) is crucial in the Ascorbate (ASC)-Glutathione (GSH) cycle, scavenging H2O2 in plant cells [53]. We found that in 6 h and 24 h, there are more up-regulated genes involved in rice seedlings after X-ray treatment. It reflects the difference in the activation time of antioxidant system of rice seedlings under carbon ion beam and X-ray radiation. In addition, our results showed that CIBs induced less genes related to antioxidant enzyme activity, which implies that the indirect damage caused by heavy ion beams is less than that caused by X-ray, which means that CIBs with a high LET cause more physiological damage to the plant compared to X-ray with a low LET. This means that high LET heavy ion beams cause less physiological damage to plants than low LET such as X-ray.

Conclusions

In this study, we discovered that radiation evoked a series of dynamic responses in rice seedlings: firstly, within 24 h, radiation induced DNA damage and activated DNA repair responses. Additionally, signal transduction processes like plant hormone signaling were activated. Finally, due to the ROS generated after radiation, the activation of plant antioxidant enzyme systems, glutathione metabolism, and other related pathways further trigger stress responses associated with MAPK and hormone pathways. Through two radiations with distinct LET, we found some common DEGs implicated in the response to radiation (such as LOC4331062, LOC4333870), thereby offering insights into the general mechanism of plant abiotic stress responses (Table S7). We hypothesize that these genes will play a role in other abiotic stress responses, but the specific mechanisms of their action will be the focus of our future research.

Furthermore, we conclude that higher LET IR like CIBs caused less indirect damage in plants, making them more suitable choice for mutation breeding research. However, due to the current research limitations, we cannot confirm the stability and heritability of these changes. Future studies should analyze genetic variations in offspring, combining the effects on rice seedlings in M1 generation with the genomic variations in subsequent generations to determine the correlations with mutations.

Data availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA014790) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

Abbreviations

IR:

Ionizing radiation

CIBs:

Carbon ion beams

FPKM:

Fragments per kilo-base per million reads

GO:

Gene ontology

KEGG:

Kyoto encyclopedia of genes and genomes

DEGs:

Differential expression genes

DSBs:

DNA double stranded breaks

SSBs:

DNA single stranded breaks

HIRFL:

Heavy Ion Research Facility In Lanzhou

JA:

Jasmonic acid

LET:

Linear energy transfer

RT-qPCR:

Real-Time Quantitative reverse transcription Polymerase Chain Reaction

BER:

Base excision repair

NER:

Nucleotide excision repair

MMR:

Mismatch repair

HR:

Homologous recombination

NHEJ:

Non-homologous end joining repair

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Acknowledgements

Part of the experimental work was carried out on the biomedical platform of the Public Technology Center of the Institute of Modern Physics, Chinese Academy of Sciences

Funding

This work was supported by the National Natural Science Foundation of China (12475357, 12135016), National Key Research and Development Program (Grant No. 2022YFD1200705), Key-Area Research and Development Program of Guangdong Province (2022B0202060006), High-efficiency Mutagenesis of New Crop Varieties Using Nuclear Technology and Demonstration, the Open Research Fund of State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China (SKL-KF202225).

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Authors and Affiliations

  1. Biophysics Group, Biomedical Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China

    Jianing Ding, Chaoli Xu, Yan Du, Xiao Liu, Jingmin Chen, Zhe Li, Jing Long, Yukun Sheng, Wenjie Jin, Dan Xu & Libin Zhou

  2. State Key Laboratory of Heavy Ion Science and Technology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China

    Libin Zhou

  3. University of Chinese Academy of Sciences, Beijing, 100049, China

    Jianing Ding, Yan Du, Xiao Liu, Jingmin Chen, Zhe Li, Jing Long, Yukun Sheng, Wenjie Jin, Dan Xu & Libin Zhou

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Contributions

J.D conducted the experiments, analyzed the data and wrote the manuscript. CL.X designed and conducted the experiments. Y.D and X.L revised the manuscript and had also contribution in processing data and image analysis. JM.C, Z.L, J.L and YK.S provided technical support for the bioinformatic analysis data and image analysis. D.X and WJ.J performed the irradiation. LB.Z coordinated and supervised the project, and corrected the manuscript. All authors were involved in the interpretation, reviewed and approved the final manuscript.

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Correspondence to Libin Zhou.

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Ding, J., Xu, C., Du, Y. et al. Deciphering the molecular transcriptomic mechanisms of carbon ion beams and X-ray on rice seedlings. BMC Genomics 26, 308 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11488-y

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