Environmental factors or treatment with agents can affect plant biological macromolecules (DNA, proteins, and lipids) in a direct or indirect manner, leading to oxidative, genotoxic, and cytotoxic effects on plant cells. These effects can cause genomic instability, mutation, carcinogenesis, or even cell death, which can negatively impact plant health and result in reduced crop yield ^[1][2][33,39]. The inability of cells to preserve the integrity of their own genome is linked to primary damage in their biomolecules. The nature of primary DNA damage is similar, regardless of it occurs in microbial, animal, or plant genomes. Whether it originates from external or internal sources, damage can be hydrolytic (cleavage of glycosidic bonds, deamination of cytosine analogs, and depurination) ^[2][39]; alkylating ^[3][40]; oxidative damage ^[4][41]; damage caused by low or high LET radiation (single and double strand breaks, SSBs and DSBs, cluster damage); UV-induced damage (photolesions); or damage caused by base analogs and intercalating agents, crosslinking agents, and protein inhibitors ^[5][6][7][42,43,44]. Plant ability to respond to DNA damage caused by stress is dependent on various factors, including their anatomy and morphology, behavioral abilities, physiological resilience, phenotypic flexibility, and the effectiveness of DNA repair mechanisms ^[7][8][9][44,45,46]. Most environmental factors have a complex mode of action and can directly affect the sugar-phosphate backbone of DNA and lead to the formation of SSBs and DSBs or necessitate metabolic activation.

Hydrolytic DNA damage can occur spontaneously or as a result of stress caused by heating, alkylation of bases, or the action of N-glycosylases ^[7][44]. Some of these changes lead to the formation of abasic (apurinic or apyrimidinic, AP) sites. In maize root tip cells, spontaneous hydrolytic DNA damage occurs at a frequency of 3.75 × 10⁵ per genome/per cell during the first 20 h of seed imbibition ^[10][47]. According to Britt ^[2][39], hydrolytic damage is a common event in the plant genome due to constant exposure to oxygen and UV light. Under stress conditions, the incidence of this type of DNA damage considerably increases.

Exposure to high temperatures can result in an accumulation of hydrolytic damage (such as deaminated cytosine and AP sites) and oxidative damage (such as 8-oxoguanine) in the plant genome ^[11][48]. However, plants have the ability to effectively cope with a significant amount of DNA damage through mechanisms that include tolerance or an enhanced repair capacity ^[12][49].

Oxidative damage in plant cells is one of the best studied phenomena. Sunlight, specifically its most energetic components (low and high LET; UV radiation), along with air pollutants such as ozone, possess enough energy to excite electrons and ionize water molecules. This results in the formation of water radiolysis products (reactive oxygen species, ROS), hydrated electrons (e_aq⁻), ionized water (H₂O⁺), hydroperoxyl radicals (HO₂•), hydroxyl radicals (•OH), hydrogen radicals (H•), reactive nitrogen species (RNS) ^[12][13][49,50], and hydrogen peroxide (H₂O₂). These events occur within a very short period of time (~10⁻⁸ s) ^[14][51]. During stress, there is typically an increase in the imbalance between the production and scavenging of ROS ^[15][16][17][52,53,54]. Recently, two pathways of ROS generation in plant cells have been identified: through plasma membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and peroxidase; or during “electron leak” in chloroplasts, mitochondria, and peroxisomes ^[18][55]. Furthermore, different types of ROS have varying levels of reactivity towards DNA. Møller et al. ^[19][56] found that among all ROS, only •OH is able to quickly react with DNA molecules. Furthermore, singlet oxygen can also react with DNA, but it primarily does so with guanine. ROS-induced damage to the genome can take many forms, including more than 100 different types of base damage (e.g., single pyrimidine and purine base lesions, inter- and intrastrand crosslinks, purine 5′, 8-cyclonucleosides, DNA–protein adducts, etc.) ^[20][57]. Extreme temperatures (frost, cold and heat stress), salinity, drought, water and nutrient imbalance, and high light intensity can trigger oxidative damage in the plant genome ^[21][58]. Accumulation of ROS can disrupt the photosynthetic system, carbohydrate metabolism, and some plant cell structures and mechanisms, regardless of whether oxidative damage is caused by endogenous processes or external agents ^[12][22][49,59].

To combat the detrimental effects of oxidative damage, plants have developed a variety of antioxidant regulatory systems, both enzymatic and non-enzymatic ^[21][58]. Furthermore, plants possess mechanisms for tolerance, such as ignoring or neglecting induced DNA damage without repair, as discussed by Roldán-Arjona and Ariza ^[12][49]. Oxidative stress is often accompanied by nitrosative stress ^[23][60]. Activation of RNS, such as NO and nitric dioxide (NO₂), as well as nonradicals, such as nitrous acid (HNO₂) and dinitrogen tetroxide (N₂O₄), when combined with superoxide radical (O₂•) can lead to disruptions in lipids, thiols, proteins, and DNA bases ^[19][24][56,61]. These signaling molecules are typically associated with the response to abiotic and biotic stresses, but they also play a crucial role in regulating various processes in plants, such as metabolism, growth and development, solute transport, autophagy, and programmed cell death (PCD) ^[25][62]. Despite their importance in plant stress response, their potential role in IR stress has not been fully explored ^[24][61]. Primary alkylated DNA lesions are a common occurrence in plant genomes, regardless of their endogenous or exogenous origin. They result from the addition of alkyl groups (such as methyl or ethyl group) to oxygen and nitrogen atoms within DNA bases and phosphodiester bonds ^[6][43]. These lesions have genotoxic, cytotoxic, or mutagenic properties because they can obstruct gene transcription and replication process within the plant genome.

Secondary damage to the genome, such as AP sites, DNA strand breaks, and interstrand crosslinks, can also occur ^[26][63]. In human genomes, the most common alkylated DNA lesions are N7-methylguanine and N3-methyladenine ^[6][27][43,64]. These types of DNA damage can block replication and transcription, leading to the formation of AP sites ^[27][28][64,65]. AP sites can be produced spontaneously or enzymatically during base excision repair (BER). In Arabidopsis, it has been suggested that different groups of endonucleases are active for the removal of AP sites ^[28][65].

The use of alkylation damage in plants is often utilized in breeding and genetic modification programs. There are different types of alkylating agents, which are classified by the number of reactive sites as monofunctional, bifunctional, and polyfunctional ^[29][66]. These agents can also be classified by their specific nucleophilic substitution reactions, either as monomolecular nucleophilic substitution (S_N1) or bimolecular nucleophilic substitution 2 (S_N2) ^[30][67]. S_N1 agents can affect both nitrogen and oxygen atoms on the bases, while S_N2 agents mainly affect ring nitrogen atoms (N1, N3 and N7) on the bases. These mutations have been utilized to improve cereal crops and medicinal plants, as well as to breed Capsicum annuum in order to obtain economically valuable traits. Examples of S_N1 alkylating agents include N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), while an example of S_N2 agents is methyl methanesulfonate (MMS) ^[31][32][68,69].

The main components of solar radiation, namely UV-B and short-wavelength UV-C, induce direct photolesions, such as cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs), as well as indirect DNA damage through the accumulation of ROS. UV-A radiation primarily causes SSBs, alkali-labile lesions, and oxidative DNA damage due to the low level of absorption by DNA ^[33][70].

Exposure to IR, both high and low LET, primarily causes base damage and direct DNA strand breaks, including SSBs and a small number of DSBs (<5%). These breaks occur randomly throughout the plant genome. Depending on the intensity of exposure, AP sites and cluster damage may also occur ^[34][71]. It is known that low doses of low LET radiation do not usually cause breaks in the DNA chain. When such breaks do occur, they are more likely to be SSBs than DSBs ^[35][72]. SSBs are less severe for the genome as they can be repaired quickly, usually within a minute, as the cell has a copy of intact DNA and can restore its structure during repair. However, if there are more than 100,000 SSBs, cell-cycle arrest may occur. In contrast, DSBs are primarily caused by the breakage of phosphodiester bonds between the sugar residues of two complementary DNA, which occur 10–20 bp apart in both DNA strands ^[36][73]. It is known that one to ten DSBs can lead to cell-cycle arrest and ultimately cell death ^[37][74]. DNA strand breaks can also be caused by other complex types of abiotic stressors. For instance, heat stress can lead to SSBs and DBSs ^[11][48], while cold stress can cause DNA damage, including DSBs in root stem cells ^[38][75].

Clustered DNA damage refers to complex DNA injury (including DSBs and non-DSBs) that occurs when there are at least two or more lesions of the DNA helix resulting from single exposure to IR or treatment with radiomimetic agents ^[39][40][76,77]. This type of damage includes harm to DNA bases, SSBs, and AP, as well as modifications to sugar residues ^[39][76]. It is believed that cluster damage mainly occurs as an indirect effect of radiation exposure and is highly dependent on the neutralization of free radicals and the structure of chromatin ^[41][42][78,79]. Research on the impacts of IR on plants offers an opportunity to study changes in regions affected by radiation pollution, such as Chernobyl, Fukushima, and the Marshall Islands, as well as in naturally radioactive areas ^[43][44][80,81], to track the adaptation process. Additionally, using radiation in a controlled environment allows radiation mutation breeding to create new plant varieties with desirable traits ^[45][82]. In addition to traditional forms of radiation, such as X-rays and gamma rays, there is growing interest in using high-energy particle radiation to enhance the quality of ornamental plants ^[46][83], and economically important crops ^[46][47][48][83,84,85], as well as for algae biofuel production ^[49][86].

Investigating the impacts of chemical agents on plants, known as plant ecotoxicology, can be challenging due to the complexity of interpreting the relationship between the dose of the agent and the observed biological effects. Basic models, such as dose–response models, can aid in understanding this relationship by showing how different doses of a chemical agent can lead to different levels of DNA damage in plants. The impact of chemicals on plants can vary depending on the type of agent, concentration, duration of exposure (acute or chronic)], and the mode of action in which the chemical acts on the plant ^[50][51][92,93]. Furthermore, the choice of plant species, testing method (in vivo or in vitro), and the way the chemical is absorbed and metabolized by the plant cells can also affect the results of ecotoxicological research and the effects of the chemical on plants.

Agents can be classified as genotoxic or non-genotoxic based on how they affect cells ^[51][93]. Genotoxic agents cause damage to DNA and proteins, and this damage can be classified as primary or secondary depending on whether or not an inflammatory response is present ^[52][94]. Primary genotoxins interact directly with cellular components and DNA and lead to the formation of harmful molecules, such as ROS/RNS. They can damage DNA directly or indirectly through the production of free radicals in the mitochondria and membrane-bound NADPH oxidases ^[52][94]. Toxicity tests, both in vivo (using live animals) and in vitro (using cell/tissue cultures of human or animal origin), aim to determine the effective dose of the agent that leads to a carcinogenic effect. Non-genotoxic or epigenetic agents primarily affect cell behavior rather than DNA. These agents include tumor promoters, endocrine modifiers, receptor mediators, immunosuppressants or elicitors, and can cause tissue-specific toxicity and inflammatory responses ^[53][95]. Most of the agents have little or no impact on plant health as plants possess mechanisms to prevent the spread of tumor cells ^[54][96]. On the other hand, some of these agents can be used to achieve specific effects in plant cells through transformation. A key characteristic of these agents is that they have a threshold dose, below which they do not produce a biological effect ^[51][93]. Similarly, plants have a threshold radiation dose of 10 mGy·d⁻¹ (417 μGy/h) before adverse effects occur ^[55][97]. Genotoxic agents directly damage DNA and chromosomes and have mutagenic effects that do not require metabolic activation. Pro-carcinogenic agents, which do require metabolic activation to cause cancer, also belong to this group. Some inorganic substances, such as metals or metalloids, can also be genotoxic. Their level and distribution in soil and water are crucial for plant growth as some are essential micronutrients ^[56][98]. However, excessive amounts could be harmful to both plants and humans. When metabolized by plants, metals/metalloids can enter the food chain and accumulate in the human body, causing harm. High concentrations of these substances in soil also lead to contamination and decreased crop yield ^[56][98]. In plant model systems, various agents cause sublethal and lethal effects ^[50][92]. These effects often have a specific dose–response relationship, which can be linear or non-linear, with a threshold or non-threshold level. As previously mentioned, non-genotoxic agents typically have a threshold dose, but it has recently been observed that some genotoxic carcinogens also have threshold doses ^[51][93].

Haber’s law, one of the earliest models used to describe the relationship between dose and response, states that toxic effects are related to both the concentration and duration of exposure. This model suggests that even small doses of a substance can have an effect, which means that exposure to low levels of a toxic substance over a long period of time can be just as harmful as exposure to a higher level for a shorter period of time. However, in reality, many toxic effects are found to be more influenced by the concentration of the substance rather than the duration of exposure ^[57][99]. This means that the amount of a toxic substance in a given environment is more crucial in determining its potential for harm than the length of time when the substance is present. It is important to note that the dose–response relationship is not always linear, and the toxic effects of a substance can also depend on the route of exposure, the organism, and other factors.

Plants, like all biological systems, are complex organisms and their response to external factors depends on various structural and behavioral factors. There are also notable differences in the way plants and animals respond to agents. According to Karban et al. ^[58][100], plants tend to have a higher threshold for agents and a lower sensitivity compared to animal models. The relationship between the amount of an agent (a drug or chemical) applied to an organism and the resulting biological response can be represented by a dose–response curve. This type of graph illustrates the relationship between the increased dose or the concentration of the agent and the corresponding increase in biological response. There are two main types of dose–response curves, namely graded and quantal. Graded dose–response curves describe the continuous relationship between increased biological response and increased dose or concentration in the single biological unit. These curves are characterized by four parameters as follows: potency (also known as the half-maximal effective concentration or dose, denoted as EC₅₀ or ED₅₀), which is the dose that produces the maximum effect; slope, which describes how steep the curve is; maximum, which is the highest level of response that can be achieved; and threshold dose, which is the minimum dose required to produce any response ^[59][101]. On the other hand, quantal dose–response curves describe the relationship between the proportion of organisms experienced or a not particular effect, known as an “all-or-nothing” phenomenon. Depending on the variation in biological response per unit dose or concentration, different types of dose–response curves can be observed. These can include monotonic curves, where the slope does not change sign; non-monotonic, where the slope changes sign ^[60][102]; and curves with no dose–response relationship (WDR), characterized by a zero slope ^[61][103]. Examples of different types of curves include linear, non-linear, threshold, sigmoidal, saturation, and U-shaped curves.

In plants, different stressors can produce various dose–response relationships depending on the specific responses and the ability or inability to overcome a particular stressor. Linear response models with or without threshold dose or concentration (linear no-threshold, LNT; linear threshold, LT) have been used for over 90 years as the standard model for assessing the risk of chemicals, radiation, and environmental agents ^[62][63][107,108]. It should be noted, however, that this model may not always be appropriate for all types of stressors and its limitations should be taken into account. The LNT model, originally developed to explain evolutionary processes ^[63][108], has become the main model for assessing the risk of IR effects used by the World Health Organization (WHO) and the Environmental Protection Agency (EPA) as the standard for human health protection. In this model, the biological effect is assumed to be proportional and have a linear or linear-quadratic curve ^[64][109]. The linear no-threshold model is based on the principle that radiation is extremely hazardous and there is no safe level of exposure. Even low doses of radiation can result in heritable genetic mutations and tumorigenesis. The target effects of radiation exposure on DNA molecules also follow this pattern ^[65][110], which is directly related to the high-dose effects of radiation on human populations. However, this does not necessarily apply to all types of stressors. Furthermore, this model does not always explain the observed short- and long-term effects of low-dose radiation ^[66][111], which have been observed in humans during space exploration, after the catastrophic nuclear disasters in Hiroshima and Nagasaki in 1945, and those affected by nuclear disasters at Chernobyl (1986) and Fukushima (2011) ^[64][109]. It is worth mentioning that alternative models, such as the hormesis or threshold model, may better explain these effects.

When evaluating the risk for plants, it is important to consider both the potential harm to the plant itself and the potential impact on human health through consumption or other means. For instance, plants grown in areas with high radiation levels or heavy pesticide use may contain dangerous levels of radionuclides or pesticides that could harm humans if consumed ^[43][67][80,112]. Furthermore, doses or concentrations that would affect humans have a greater impact on plants because they have a higher tolerance for radiation and chemical exposure. It should also be noted that various factors, such as the type of agent, the exposure level, the extent of contact with the plant, the accumulation in the plant, the distribution in different plant parts, and the plant ability to neutralize the agent, are crucial in determining the impact. It is also important to consider the context in which the exposure takes place and the specific plant species and variety being studied.

The wide variety of plant stressors leads to a number of biological effects. Ideally, stressors only affect specific targets, such as the DNA molecule, resulting in targeted effects that can be seen within one or two generations. However, in reality, organism response is more complex and off-target effects, which are not fully understood, are also observed ^[68][113]. Despite limited research in this area, radiation exposure often leads to such effects in animal and human model systems, and similar effects are seen in plants due to various environmental stressors. The known off-target effects of radiation exposure, such as signal-mediated effects, stress-induced genomic instability, transgenerational effects, sensitivity to low doses, biphasic response or hormesis, and others, also occur in the plant genome under environmental stress as outlined by the study of Joiner ^[69][114]. More research is needed to fully understand the extent and mechanisms of these off-target effects in plants.

4. Low-Dose Hyper-Radiosensitivity and Radioresistance

The concept of low-dose hyper-radiosensitivity is well-established in mammalian systems, where cells display resistance to high single doses of radiation but show sensitivity to small single doses. This is typified by a limited number of exposures, such as high and low LET IR and chemotherapy drugs ^[69][114]. However, experiments on low-dose hyper-radiosensitivity in plant models have not been adequately described ^[69][114]. Eriksson ^[70][188] is one of the few researchers to do so, reporting this phenomenon in irradiated maize plants after exposure to a dose of 50 cGy where the frequency of mutation induction and lethality in pollen grains was higher than the spontaneous mutation rate. The differences in radiation response at low and high doses of radiation are thought to be due to the different sensitivity of the cell-cycle phases ^[71][189]. Further research is needed to fully understand the mechanisms underlying low-dose hyper-radiosensitivity in plant models and to examine the implications of this phenomenon for plant breeding and crop production.

5. Biphasic Dose–Response Effects in Plants: Hormesis, Stress-Induced Priming, and Adaptive Response

Although the existence of biphasic dose–response effects in plants has been known since Darwin’s time, it has long been incorrectly associated with homeopathy, causing a stagnation in its study. Another reason for the lack of sufficient knowledge is that this phenomenon has been studied by different disciplines. For many years, it was rejected by governmental regulatory agencies because it contradicts the established dose–response approach to risk assessment ^[72][190]. Recently, Calabrese and Agathokleous ^[72][190] reported that there are over 30 different terms used to describe the biphasic dose–response model, including U-shaped, adaptive responses, hormesis, priming, preconditioning, and others, which all describe different aspects of the same phenomenon ^[73][191]. In plant studies, various terms have been employed based on the type of stressors. For instance, Ancel and Lallemand ^[74][192] used the term “preconditioning” to refer to this phenomenon in plants following X-ray irradiation. As noted in the review by Calabrese and Baldwin ^[75][193], the concept of “chemical hormesis” can be traced back to the studies from the late 19th century, which demonstrate the stimulatory effects of sodium hypochlorite on seed germination and the influence of different metals on root growth. In the mid-1970s, the term “adaptive response” was utilized to describe the same phenomenon that occurs after chemical mutagens, and later in the 1980s, it was also used for IR ^[76][194]. When defense mechanisms were activated as a result of pathogens, arthropod attacks, or adverse environmental conditions, the term “defense priming” was introduced ^[77][78][195,196]. In recent years, this phenomenon has gained increased attention, leading to its deeper understanding. Hormesis is considered a quantitative estimate of biological plasticity ^[79][197]. The basis of hormetic response is prior exposure to a low dose or concentration of a stress trigger (“priming” stress), which can reduce the toxic effects of subsequent exposure of a higher dose or concentration (“challenge” stress) of the same or a different stress trigger ^[76][80][81][194,198,199]. The hormetic dose–response curve is often depicted as a U-shaped or J-shaped curve, with the main features being the hormetic stimulatory zone (HSZ) with subinhibitory doses; the maximal stimulatory dose (MSD), which is the percentage change from the control dose (usually <200% of control response) ^[82][200]; and the selection of the no observable adverse effects level (NOAEL), or the zero equivalent point (ZEP) or thresholds, followed by inhibitory doses where adverse effects are observed ^[83][84][201,202]. The effects of hormesis on organisms can be either harmful, known as distress, or beneficial, known as eustress ^[84][85][202,203]. In plants, there are two main types of hormetic models, namely inverted U-shaped and U-shaped. The inverted U-shaped curve describes a response in which low dose increases and high dose decreases plant growth and photosynthesis parameters, genotoxicity, and mutagenesis ^[86][204]. The U-shaped curve, on the other hand, shows a reduction in adverse effects at low doses and an enhancement of adverse effects at high doses, as can be observed in defense mechanisms such as activities of the major scavenging enzymes, such as ascorbate peroxidase (APX), guaiacol peroxidase (GPX), superoxide radicals, endo-proteinase isoenzymes, carbonyl and malondialdehyde groups, etc. ^[86][204]. A third type of hormesis dose–response curve with two dents has also been proposed, but it is specific to plants and there is limited evidence of its existence. This model has been observed in different plants under heavy metal stress ^[87][88][205,206]. The scientific literature is abundant with research on the hormesis behavior of plants, which is triggered by various stress factors. These effects are observed at different levels of biological organization, including cells, organs, organisms, and communities ^[62][107]. Hormetic responses have been observed in plants following exposure to a wide range of agents that affect plant growth and development, such as macro- and micronutrients ^[89][90][207,208], biostimulants ^[91][209], herbicides and fungicides ^[92][93][94][210,211,212], heavy metals and metal ions, nanoparticles ^{[86][95][96][97][98]}[204,213,214,215,216], temperature ^[82][200], phytohormones ^[99][217], heat stress ^[100][218], light ^[101][219], and pathogens ^[77][195]. A growing body of research has shown that both IR and non-IR exposure can have hormetic effects on plants. In general, hormesis is associated with pretreatment of plants with relatively weak exposure (called conditioning clastogenic dose), which increases their resistance to radiation, followed by exposure to higher doses (or challenge dose) of the agents some hours later ^[102][220]. Hormesis can be induced by both low and high doses of radiation ^{[103][104][105]}[221,222,223]. Typically, radiation hormesis in plants has a positive effect, resulting in increased germination, growth rate, height, weight, pigment content, flowering, fertility, accelerated development, and increased radiation resistance. The degree of hormesis depends on the genetic characteristics of the seeds or plant, moisture of the seeds, type of low-dose radiation, and duration of irradiation ^[106][107][224,225]. The adaptive response triggered by hormesis includes both short-term mechanisms, such as the use of existing proteins, and long-term mechanisms, such as the expression of genes encoding specific enzyme systems. Activation of HSPs, proteasomes, and kinase cascades can also occur ^[107][108][225,226]. During hormesis, several mechanisms are activated in plants, including the detoxification of ROS through increased levels of ABA, followed by increased levels of H₂O₂, activation of DNA repair mechanisms, removal of damaged cells through apoptosis, alteration of nitrogen metabolism, and stimulation of immune response ^{[90][104][108][109][110]}[208,222,226,227,228]. The hormetic part of the adaptive response is associated with permanent genetic or epigenetic changes ^[111][229]. Recent studies suggest that epigenetic mechanisms play a role in plant adaptation and generation of transgenerational memories to stress ^[112][230]. It is worth mentioning that the complexity of hormesis requires additional research to fully comprehend its underlying mechanisms.