The response profile to chronic radiation exposure based on the transcriptome analysis of Scots pine from Chernobyl affected zone

Environmental Pollution 250 (2019) 618e626

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The response profile to chronic radiation exposure based on the transcriptome analysis of Scots pine from Chernobyl affected zone* Gustavo T. Duarte a, b, Polina Yu. Volkova a, Stanislav A. Geras'kin a, * a b

Russian Institute of Radiology and Agroecology, 249032, Obninsk, Russia Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Universit
e Paris-Saclay, 78000, Versailles, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 December 2018 Received in revised form 27 March 2019 Accepted 13 April 2019 Available online 21 April 2019

Radioactive contamination of the natural areas is one of the most long-lasting anthropogenic impacts on the environment. Scots pine (Pinus sylvestris L.) is a promising organism for radiation-related research because of its high radiosensitivity, but the genome size of Pinacea species has imposed obstacles for high-throughput studies so far. In this work, we conducted the analysis of the de novo assembled transcriptome of Scots pine populations growing in the Chernobyl-affected zone, which is still today contaminated with radionuclides because of the accident at the nuclear power plant in 1986. The transcriptome profiles indicate a clear pattern of adaptive stress response, which seems to be dosedependent. The transcriptional response indicates a continuous modulation of the cellular redox system, enhanced expression of chaperones and histones, along with the control of ions balance. Interestingly, the activity of transposable element families is inversely correlated to the exposure levels to radiation. These adaptive responses, which are triggered by radiation doses 30 times lower than the one accepted as a safe for biota species by international regulations, suggest that the environmental management in radiation protection should be reviewed. © 2019 Elsevier Ltd. All rights reserved.

Keywords: RNA-Seq Chronic radiation Pinus sylvestris SNP Transposons

1. Introduction Abiotic factors, such as water availability, extreme temperatures, or pollutants, alongside with biotic factors, impose continuous challenges to the ability of plants to adapt. The concern about the adaptability of plants increasingly becomes important because of the rapidly changing climate, both in terms of the conservation of natural populations and the continued exploration of economically important species (Des Marais et al., 2013). While the environmental awareness is increasing and improving the management of natural areas, radiation disasters such as Fukushima accident in 2011 show that studies about the long-term consequences of radiation exposure are still lagging behind. Ionizing radiation can cause damage to proteins and cellular structures, disrupting signal transduction pathways and culminating on cell death (Jan et al., 2012; Einor et al., 2016). The mutations caused by ionizing radiation in DNA molecules have long

*

This paper has been recommended for acceptance by Dr. Yong Sik Ok. * Corresponding author. 249032, RIRAE, Kievskoe shosse, 109 km, Obninsk, Kaluga region, Russia. E-mail address: [email protected] (S.A. Geras'kin). https://doi.org/10.1016/j.envpol.2019.04.064 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

been discussed as an evolutionary factor (Esnault et al., 2010; Byrne et al., 2014; Møller and Mousseau, 2016). These effects are caused by direct influence of radiation on DNA and by the reactive oxygen species (ROS), which are overproduced as a consequence of the radiolysis of water by the highly energetic electrons (Riley, 1994). Interestingly, while plants have diverse strategies for minimizing the effects of adverse conditions (e.g., specific alterations in metabolic pathways, accumulation of osmoprotectants, and modifications of gene expression profiles), a converging point during most of plants stress responses is also ROS generation (Sewelam et al., 2016; Zhu, 2016). Although much has been done on the simulation of stress conditions by time-limited laboratory experiments, field conditions studies are not easily reproducible. Indeed, while stress-promoted acute responses are well-documented, stress is often a recurrent €mke and B€ or chronic condition in natural environments (La aurle, 2017). In this context, among the anthropogenic factors chronically impacting natural populations, the elevated level of radiation is one of the most long-lasting, but still poorly studied. While the consequences of transient radiation exposure to plants have been experimentally addressed (Scheibe et al., 2005; Kovalchuck et al., 2007; Esnault et al., 2010; Jan et al., 2012), this knowledge on

G.T. Duarte et al. / Environmental Pollution 250 (2019) 618e626

wild populations is still limited (Geras'kin, 2016) either due to the lack of sufficient natural areas or the lack of concern. The study of the consequences of chronic radiation exposure relies on regions showing high natural radioactivity, or on territories contaminated by radiation accidents. In this context, the Chernobyl nuclear power plant (CNPP) disaster, which released 1.85
1018 Bq of fission products (IAEA, 2006), was one of the largest nuclear incidents in history. The radioactive contamination by the CNPP disaster affected a broad range of ecosystems, what in turn made the Chernobyl exclusion zone a unique source of information for accessing the effects of ionizing radiation on the abundance, distribution, life history, and mutation rates on different species (Geras'kin et al., 2008; Galv
an et al., 2014; Møller and Mousseau, 2015; Geras'kin, 2016; Volkova et al., 2017). Nevertheless, a consensus about the effects of long-term chronic low-dose irradiation on biota at
chignac et al., 2016). In this Chernobyl has not been reached yet (Bre respect, Scots pine (Pinus sylvestris L.) is a promising organism for radiation-related studies because of its high radiosensitivity (ICRP, 2008; Geras'kin et al., 2018), and can be used to address the consequences of chronic low-dose radiation exposure. As one of the most widespread tree species across Europe and Northern Asia, Scots pine is a reference for radiation protection of biota (ICRP, 2008), while it also has high economic value for wood forestry. In this context, intending to gain insight into how plants are able to withstand chronic radiation exposure, we evaluated the transcriptome profiles of individuals from four Scots pine populations, two of which belonging to the Chernobyl exclusion zone. 2. Materials and methods 2.1. Sampling and sample plots The sample plots are located in the Bryansk region of Russian Federation and in the Gomel region of Belarus Republic. The reference plot (Ref, 52º460 13.4400 N 32º390 34.7900 E, 218 km to CNPP) and the low-contaminated Zabor'e plot (Zab, 53º5011.3400 N 31º42010.3200 E, 232.8 km to CNPP) are both located in the Bryansk region. The highly contaminated Kulazhin (Kul, 51º330 42.800 N 30º10011.000 E, 19.8 km to CNPP) and Masany (Mas, 51º300 43.000 N 30º01007.200 E, 14.7 km to CNPP) plots are situated in the Gomel region, in the Chernobyl exclusion zone. We selected trees between 20 and 25 years old to perform the transcriptome analysis, meaning that they are of the first generation after the CNPP accident. These trees have been chronically exposed to radiation during their lifetime, but not to the acute irradiation period after the disaster in 1986. Each biological sample consisted of pine needles collected from the four cardinal directions of the tree crown (pool of 12 needles per individual) from three different individuals (total of 36 needles per sample). Three replicates for each biological sample were collected at each location, and immediately frozen in liquid nitrogen. At all sample plots, pine trees are dominant in the phytocenosis and the samples were collected during similar weather conditions on May 2016. At each sample plot, soil samples were collected for the evaluation of their properties, and of the radioactive and chemical contaminations. The soil was collected from the corners of a rectangular area (1
2 m) around each individual that was selected for the needles sampling, and from the central position where the tree was located. These five soil samples were pooled at three different depths (0e5 cm, 5e10 cm, 10e15 cm). Next, they were airdried at room temperature in laboratory and 1 mm sieved. The following basic physicochemical properties were analysed in accordance with the ISO standard for soil quality (ISO/TC 190): pH, hydrolytic activity, K, P, and Ca contents, cation exchange capacity,

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and humus content. 2.2. Heavy metal and radioactive contamination Total concentrations of heavy metals (Cd, Cu, Co, Ni, Cr, Mn, Pb, Zn) were measured in the soil samples by treatments with a mixture of HNO3, HCl, and HF, as previously described (Geras'kin et al., 2011). Concentrations of heavy metals in solution were determined using plasma optical emission spectrometer (ICP-OES, Varian, Australia-USA) in accordance with the ISO 11047 standard (ISO 11047, 1998). The quantification limit was 1e10 mg L1. The exposure dose rates at the sample plots were estimated using a gamma dosimeter DRG-01T (NPP Doza, Russia). The dosimeter allows dose rate estimation between 0.01 mR h1 and 9.99 R h1 (error < 15%). The dose rates were measured for 5 times at 1 m above the ground under each tree that was sampled. The assessment of 137Cs (the main g-emitting radionuclide at the territories contaminated by the Chernobyl accident) was done with a gamma-spectrometer CANBERRA Packard (USA) with a coaxial semiconductor Ge(Li) detector and an extended energy range. The range of g-radiation energy measurements was 40e10000 keV. The relative error of activity was 2e7%, depending on the sample. Given that a large number of needles are necessary for the estimation of the annual dose received by pine crowns, we used pine cones for preserving the trees. The 137Cs activity concentrations were measured by g-spectrometry. To determine 90Sr activity concentration in cones, they were heated to ashes at 600 С, following extraction of 90Sr by double treatment with boiling solution of 6 М HCl. The 90Sr content in the samples was estimated using the equation:

A90Sr ¼

A90Y WSr
WY
m

where A90Sr e

Sr activity concentration in the sample, Bq kg1;

90

A90Y e 90Y activity in the sample, Bq; WSr e Sr chemical yield; WY e Y chemical yield; m e air-dried mass of the sample, kg (±30% error). After measurement for 1800 s, the minimum detectable limit was 0.05 Bq. 2.3. Dosimetric model To estimate the doses accumulated by pine crowns, we used the dosimetric model developed for the generative organs of pine. The g-radiation dose rates were calculated based on the active concentrations of 137Cs in soil according to our previous work (Geras'kin et al., 2011). b-radiation dose rates were estimated according to the specific activities of 137Cs and 90Sr (90Y) in pine cones, using formula for a spherical source. The size of pine cones significantly exceeds the mean free path of a-particles, what allows the use of an infinite source according to the following formula: ‘

Da ¼ K
E a
Qa a e energy of an a-particle per decay; where E Qa e concentration of a-emitters; K e numerical coefficient. The concentrations of transuranic radionuclides (238241Pu and

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241 Am) in pine cones were estimated based on their coefficients of accumulation (Parfenov, 1995). As input data, the mean values of pollution density at the sample plots (ARPA, 2009) were used.

grouped according to the levels of radioactive contamination (Mas/ Kul, Ref/Zab), suggesting that the transcript expression patterns correlate with the radiation exposure. The comparison of Mas, Kul, and Zab populations against the reference plot revealed a total of 225, 278 and 69 differentially expressed (DE) transcripts, respectively (p < 0.001; log2FC > j1j). Fig. 1 depicts the shared up- and down-regulated transcripts among the three populations. Few transcripts are commonly regulated among the three contaminated areas (five down-regulated and two up-regulated) (Table 4). But as expected, Mas and Kul, which are the most contaminated plots, share the higher overlap of DE elements (Fig. 1). Among these genes, only the S-type anion channel (SLAC1_ARATH) was reported to respond to acute radiation exposure in Arabidopsis thaliana, although being up-regulated (Table S3). Indeed, few of the DE transcripts identified in the chronically irradiated samples are responsive to acute irradiation. Most of the transcripts that overlap between these two conditions are marked by an antagonistic expression pattern (Table S3). Zab population faces until today a relatively high level of g-radiation exposure (Table 1), which is equivalent to that observed for Mas plot. Nevertheless, in this population, few DE transcripts were found (Fig. 1, Table S3). It is possible that a certain dose threshold, as represented by the annual doses sum (Table 1), may be necessary for triggering chronic radiation response as observed for Mas and Kul plots. Alternatively, chronic g-radiation alone may not suffice to induce such a response. For understanding the biological meaning of the DE transcripts in the context of our study, they were classified according to the Gene Ontology (GO) terms, following the analysis of enriched GO categories for each population (Fig. S2, Fig. S3). The trend observed for the response to chronic radiation exposure is the control of processes related to cell homeostasis. However, unlike acute radiation/oxidative stress responses, the system seems not to solely dump the accumulation of free radicals based on enzymatic reactions. Given that ROS are also signalling molecules, it is plausible to hypothesize that their complete long-term repression would lead to growth and development deficits. Instead, evidence points to the modulation of ROS generating processes along with the control of the antioxidant system, histones, chaperones, and of ions balance to attenuate the cellular damage. No enriched GO category was found for Zab plot. Between Mas and Kul plots, three are the common tendencies among the upregulated enriched transcripts: involvement of cell membranous organelles, regulation of chromatin/nucleosome assembly and organization, and control of light-related processes (i.e. photosynthesis control and response to light intensity, respectively) (Fig. S2). Light-related processes are a source of ROS, which accumulation is also one of the read-outs of the response of cells to radiation exposure (Volkova et al., 2017). Indeed, ROS-related responses are enriched in Kul plot, as for instance response to hydrogen peroxide and hydrogen peroxide transmembrane transport (Fig. S2a). In Mas plot it is observed the enrichment for mechanisms that counteract

2.4. RNA sequencing and bioinformatics analyses Detailed description of the RNA sequencing procedures and bioinformatics analyses are given as Supporting Information, and in the publication companion to this work (Duarte et al., forthcoming). 3. Results 3.1. Radionuclide and heavy metal contamination of the sample plots To assess the chronic radioactive stress conditions to which the sample plots are exposed, we measured the active concentrations of radionuclides in soil and in cones, and the contribution of the different types of radiation to the annual dose (Table 1). The maximal annual doses were found, as expected, at the plots in the vicinity of the CNPP (Kul and Mas). These values, although considered to be safe for biota species, are more than one hundred times higher than the radiation limit for humans (UNSCEAR, 2008). Zab, one of the most contaminated areas in the Russian Federation, has radiation levels three times lower than Kul or Mas. The Ref plot showed background values. The basic soil properties of the four sample plots are typical for this region and similar to each other (Kovda and Zyrin, 1981) (Table 2). The soil properties vary in a range typical for the studied region, where Scots pine often represents a dominant forest species (Rysin and Savelieva, 2008). The main parameter of difference between soils of Russian (Ref and Zab) and Belarus (Kul and Mas) plots are the contents of accessible potassium and phosphorus. Given that the individuals are acclimated to their conditions and that Scots pine requires low levels of phosphorus and potassium for proper development (Ingestad, 1979), no transcriptional changes triggered by differences on the levels of these two nutrients were expected. Indeed, according to morphological studies of pine needles from the experimental populations, no signs of nutrimental deficiency were found (unpublished data). To exclude a coaction of other chemical stressors, the concentrations of heavy metals in the soil of each plot were estimated. Heavy metal contents do not exceed the international permissible values (Nicholson and Chambers, 2007) and do not essentially differ among each of the plots (Table 3). 3.2. Chronic radiation-induced transcriptome changes The Scots pine transcriptome was represented by 98870 unique transcripts, of which 76.5% were successfully annotated. The clustering analysis indicated a strong correlation between the replicates of each population (Fig. S1). Moreover, the sample plots were Table 1 Radioactive contamination on the sample plots and the annual dose absorbed by trees. Plot Exposure dose rate (mR h1)

Contamination by radionuclides Soil 137

Ref Zab Kul Mas

11.5 163 359 263

Annual dose in 2016 (mGy) 2

Cones

Cs (Bq kg1)

156 ± 19.8 46200 ± 3243 66300 ± 820 41700 ± 330

90

12.6 ± 3.8 1420 ± 176 5470 ± 666 14000 ± 1700

Sr (Bq kg1)

0.79 ± 1.3 48.69 ± 3.7 1050 ± 12.6 4246 ± 40

Contamination density (kBq m

)

238,239,240

241

e e 10e20 20e40

Pu

241

Pu

e e 250e500 500e1000

Am

e e 4e20 >100

Dg

Db

Da

Dsum

0.22 38 62.6 37.4

0.006 0.57 4 13.5

0 0 35.1 77.6

0.23 38.6 101.7 128.5

Note. Exposure dose rates (g-radiation) were measured at each plot with a dosimeter 1 m above the ground. The annual doses were calculated with the dosimetric model detailed in the Methods.

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Table 2 Soil properties of the four sample plots. Sample plot

Reference Zabor'e Kulazhin Masany

Soil properties pHKCl

Hydrolytic acidity (mg-equ 100 g 1)

K2O mob. (mg kg1)

P2O5 mob. (mg kg1)

Cation exchange capacity (mg-equ 100 g1)

Ca mob. (mg-que 100 g1)

Humus (%)

4 4.6 4.1 4.1

2.8 10.3 18.6 20.4

108.7 139 16 15.1

244.2 133 42.1 38.4

0.82 0.95 0.09 0.08

1.3 1.2 1.7 2.6

0.8 2.5 0.7 0.7

Note. Mobile form (Mob); milligram equivalent (mg-equ).

Table 3 Heavy metal content in the soil of the four sample plots. Sample plot

Reference Zabor'e Kulazhin Masany

Element concentration (mg kg1) Cd

Cu

Co

Cr

Mn

Ni

Pb

Zn

0.06 ± 0.01 0.11 ± 0.01 0.20 ± 0.07 0.26 ± 0.14

3.4 ± 2.6 1.6 ± 0.1 5.7 ± 0.8 5.4 ± 1.8

1.7 ± 0.2 1.2 ± 0.1 0.8 ± 0.1 0.8 ± 0.3

6.4 ± 0.6 5.3 ± 0.1 2.6 ± 1.2 4.7 ± 0.2

233.9 ± 2.9 246.2 ± 37.0 87.7 ± 14.9 101.3 ± 4.5

2.1 ± 0.1 2.7 ± 0.1 2.5 ± 1.3 4.8 ± 1.0

6.9 ± 0.4 7.2 ± 0.1 3.6 ± 0.8 3.9 ± 0.9

18.8 ± 3.4 18.7 ± 0.2 9.4 ± 1.3 6.8 ± 1.9

Fig. 1. Venn diagrams indicating the unique and shared transcripts differentially expressed under chronic radiation exposure conditions in Masany (Mas), Kulazhin (Kul), and Zabor'e (Zab) plots in comparison to the reference (Ref). (A) down-regulated elements; (B) up-regulated elements.

ROS activity (Fig. S2c). For example, the oxaloacetate decarboxylase activity is related to the conversion of oxaloacetate into pyruvate, a ROS scavenger (Kładna et al., 2015), while malate dehydrogenase converts oxaloacetate to malate, described to play a role on the poising mechanism for the cell redox levels (Scheibe et al., 2005). Another interesting output observed in Kul plot is the control of iron-sulphur cluster proteins (Fig. S2c), which have a central role in photosynthesis, genome stability, electron transfer, and oxidationreduction reactions (Zhang, 2015). The overlap of functions is more evident between Mas and Kul plots down-regulated transcripts (Fig. S3). Several of the enriched GO terms are associated with the balance of ions and their

transport, but also transcription regulation and kinase binding activities, responses to ROS, cadmium, ozone, carbon dioxide and hormones (e.g. auxin and ABA), and the regulation of water homeostasis and stomata movement (Fig. S3). These responses are aligned with the function of the most deregulated transcripts (Table 4). Considering that the chronic exposure to radiation may lead to outbalance of the ROS levels (Einor et al., 2016; Volkova et al., 2017), the photosynthesis rate, as a ROS source, may be under fine tuning through the control of gas exchanges via stomata. Cadmium exposure is also known to trigger generation of ROS in plant cells (Gill and Tuteja, 2011), thus the presence of cadmiumresponsive genes may represent a convergence of response. Specific enriched GO terms are also observed in Mas or Kul as, for instance, telomere maintenance and control of sugar levels, respectively, and which may represent alternative adaptation strategies. While telomere attrition was suggested to be caused by high ROS levels in mammals (Colavitti and Finkel, 2005), sugars might act as osmoprotectants (Slama et al., 2015). Next, to define the transcriptional response profile to the chronic radiation exposure, we focused on the analysis of the pathways under gene expression control that overlap between the most contaminated plots, Mas and Kul. 3.2.1. Histones Histones are the core proteins of the nucleosomes, thus controlling the access to the information encoded in the DNA. Several transcripts for histones are up-regulated in both Mas and Kul plots. Two transcripts for H1 and one for H4 are commonly regulated between these two populations. In Kul it is also observed the

Table 4 Overlapping transcripts among the three radioactively contaminated plots. Transcript ID

UniProt ID

Function

Description

log2 FC Mas

Kul

Zab

evgTrinTRINITY_DN60304_c1_g1_i1 evgSOAPd26480654005 evgSOAPd244693109749 evgSOAPd25748839369 evgSOAPd241551103465 evgTrinTRINITY_DN60799_c3_g2_i4 evgSOAPd203292154205

WUN1_SOLTU HY5_SOLLC CIPK5_ORYSJ CIPKI_ORYSJ SLAC1_ARATH P2B11_ARATH e

Cell death Photomorphogenesis Abscisic acid signalling

Wound-induced protein 1 Transcription factor HY5 CBL-interacting protein kinase 20 CBL-interacting protein kinase 10 Guard cell S-type anion channel SLAC1 F-box protein PP2-B11 e

2.8 2.2 1.3 3.2 1.6 4.0 2.0

2.4 1.7 2.3 4.7 2.0 4.2 2.3

1.8 1.1 1.3 1.9 1.7 3.1 1.5

Stomatal closure Protein ubiquitination Uncharacterized protein

Note. The expression values are given as log2 Fold Change in comparison to the Ref population.

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induction of a H3.2 histone, while Mas individuals show the upregulation of eight other transcripts encoding for H1, H2A.1, H2A.6 and H4 (Table S3). It is interesting to note that the expression of canonical histones (H2A, H2B, H3, and H4) is usually related to the S phase of the cell cycle (Jiang and Berger, 2017). However, no cell cycle-related genes were identified among the DE transcripts, meaning that the control of the expression of histone genes must be linked to the adaptation process to chronic radiation. 3.2.2. Heat shock proteins Heat shock proteins (HSPs) are molecular chaperones which are involved in diverse adaptive responses to adverse conditions, including oxidative stress (Kotak et al., 2007). Differentially expressed transcripts encoding for HSPs are among the most represented in both Mas and Kul transcriptomes, all of them being upregulated, suggesting that these chaperones may play an important role on the adaptation to chronic radiation exposure. Six different HSPs are commonly regulated between Mas and Kul (HS174_ARATH, HS17C_ARATH, HS23C_OXYRB, HS22C_SOYBN, HSP22_ARATH, and HS901_ARATH; Table S3). In addition, Mas and Kul individuals show, respectively, one and eleven other up-regulated transcripts encoding for HSPs (Table S3). 3.2.3. Ion flux, ABA-related responses and stomata control The up-regulation of transcripts for HSPs along with the downregulation of those encoding for CBL-interacting protein kinases (CIPKs) and for the guard cell S-type anion channel 1 (SLAC1) (Table 4, Table S3) is a signature presented by ABA-promoted responses, as revealed by an analysis with Genevestigator (Hruz et al., 2008) using the respective A. thaliana homologues (data not shown). Although response to ABA is among the enriched GO terms (Fig. S3), no transcript related to ABA biosynthesis or catabolism was identified. Conversely, two transcripts in Kul plot belonging to ABA-induced genes, namely RD21B_ARATH and RD22_ARATH, are repressed (Table S3). This observation suggests that the expression control of ABA-responsive transcripts occurs by a different pathway rather than the modulation of the ABA levels. Several pieces of evidence indicate that the control of stomata movement is important for the response to chronic irradiation. CIPKs are the main target of calcium sensors, mediating Ca2þ signal transduction for adaptive responses to stress conditions (Bender et al., 2018). Ca2þ also mediates stomata closure by activating the S-type anion channels and suppressing the inward Kþ channels (Laanemets et al., 2013). In A. thaliana, the S-type anion channel SLAC1 was reported to be a negative regulator of stomata opening by controlling Kþ flux (Zhang et al., 2016). While the control of ion fluxes is in accordance with the GO enrichment analysis (Fig. S2, Fig. S3), the down-regulation of transcripts encoding for CIPKs and SLAC1 proteins in Kul, Mas and also Zab plots (Table 4, Table S3) suggests the maintenance of open stomata. This idea is further supported by the down-regulation of the negative regulator of stomata closure IMB1_ARATH (Luo et al., 2013), and the induction of LSU4_ARATH, which was described to be required for stomata closure depending on ROS in A. thaliana (Garcia-Molina et al., 2017) (Table S3). 3.2.4. Modulation of reactive oxygen species Glutathione is one of the most important non-enzymatic antioxidants in plant cells (Das and Roychoudhury, 2014), being a pivotal component of the glutathione-ascorbate cycle that controls hydrogen peroxide levels. Our data suggest that the levels of glutathione in chronically irradiated pine trees are being modulated by the control of its precursors as, for instance, glutamine. The L-glutamine/glutamate transporter WTR14_ARATH and the glutathione S-transferase GSTUJ_ARATH are both up-regulated in

Mas and Kul plots (Table S3). Moreover, WTR14_ARATH has also been reported to accumulate under hypoxia, a source of ROS (Liu et al., 2005). In Kul population, the transcript for gammaglutamylcyclotransferase GCT21_ARATH, which is related to glutamine homeostasis, is also up-regulated (Table S3). A probable phospholipid hydroperoxide glutathione peroxidase (GPX4_MESCR) is also induced in Kul plot, which enzyme has been described to play a major role on the protection of biomembranes (Jain and Bhatla, 2014) (Table S3). Besides glutathione, thioredoxins, inositol and its derivatives are important molecules controlling the redox status of target proteins, protecting the cells against oxidative stress (Vieira Dos Santos and Rey, 2006; Saxena et al., 2013). Transcriptional evidence suggests a direct control over the level of these molecules, as represented by the up-regulation of TRXH_PICMA (Mas and Kul plots), AAED1_ARATH and IMPP_MESCR (both in Kul) (Table S3). Supporting the active role of thioredoxins in response to chronic irradiation, stressresponsive genes which are targets of these molecules are also up-regulated in Mas (RPI3_ARATH and POP3_ARATH) and Kul (AL2B4_ARATH; Table S3). Moreover, the accumulation of the transcript for MSRB1_ORYSJ is also enhanced. This transcript encodes for a peptide methionine sulfoxide reductase which acts against oxidative stress by the reduction of methionine sulfoxide to methionine in oxidized proteins (Table S3). Curiously, transcripts related to the biosynthesis of ascorbic acid (vitamin C; GME1_ORYSJ and GME1_ORYS) and pyridoxal phosphate (vitamin B6; Y1015_ARATH), both of which described as antioxidants (TambascoStudart et al., 2005; Watanabe et al., 2006), are repressed (Table S3). While the antioxidant system components usually act as ROSscavengers, another strategy for keeping the system at homeostasis must occur via the balance of NAD(P)H-related elements, which take part on electron transport chains (Scheibe et al., 2005). Accordingly, SRC2_ARATH, UBA2B_ARATH, PROD2_ARATH, PCKA_ARATH, and GLPT1_ARATH, are all down-regulated in Mas and/or Kul plots (Table S3). SRC2_ARATH encodes for an activator of the Ca2þ-dependent NADPH oxidase AtRbohF, which mediates ROS production during stress responses (Kawarazaki et al., 2013). UBA2B_ARATH (UBP1-associated protein 2B), PROD2_ARATH (proline dehydrogenase 2), and PCKA_ARATH (phosphoenolpyruvate carboxykinase) have all been described to trigger ROS accumulation (Dizengremel et al., 2012; Na et al., 2015; Nagano et al., 2017). 3.3. Transposable elements activity Since it has been shown that ionizing radiation can activate TEs in Drosophila melanogaster (Kravets et al., 2010) and yeast (Sacerdot et al., 2005), we decided to investigate the activity of TEs in plants under chronic radiation exposure. A total of 991 unique contigs matching to TEs were found among Ref, Kul, and Zab plots (Mas was not used for this analysis, as detailed in the Methods). Copia, Gypsy, IFG7, PtAngelina, PtAppalachian, and PtCumberland were the families with the highest number of active elements (Fig. 2). Exposure to radiation clearly activates the TEs in Kul and Zab, but interestingly in a different way. In comparison to the Ref, the lowcontaminated Zab plot had more elements with induced activity than the highly contaminated Kul plot (Fig. 2a). In contrast, more repressed TEs were found in Kul than in Zab population (Fig. 2b). This result raises two hypotheses. First, as observed for the control of gene expression regarding responses at the low- or highlycontaminated plots, a certain dose threshold may be necessary for switching the control of TEs activity. Second, considering that Zab population is mainly under g-radiation exposure, while Kul plot also registers high levels of a- and b-radiation, the radionuclide composition may have an important role on the activation and control of the TEs (Table 1).

G.T. Duarte et al. / Environmental Pollution 250 (2019) 618e626

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Fig. 2. Relative activity of TEs in comparison to the Ref plot of the most represented families in the regions highly (Kul) and lowly (Zab) contaminated by radionuclides. (A) Number of TEs induced by radiation exposure. (B) Number of TEs repressed by radiation exposure. The numbers of TEs are in log2 scale.

3.4. Single-nucleotide polymorphisms Given that chronic radiation exposure is capable of changing the genetic structure of populations (Volkova et al., 2017), we evaluated the occurrence of single-nucleotide polymorphisms (SNPs) which could be related to radiation exposure in each location. Only variations that were not observed in the Ref population were retained. Moreover, identical polymorphisms observed in all samples from the same location were excluded, as they most likely represent a population polymorphism rather than a radiation-induced one. A total of 6717, 2949, and 5646 SNPs were identified in Kul, Mas, and Zab plots, respectively. The number of SNPs for Mas is, however, underrepresented, by reason of only two samples being available for the analysis. Considering that radiation-induced mutations must occur at random, it was surprising the number of SNPs occurring at the same positions, as shown by the overlap analysis (Fig. S4). It is interesting to speculate if these mutations could be occurring on specific genes according to their function. To investigate this hypothesis, GO enrichment analysis was run on the 608 unique transcripts that harbour the 766 overlapping SNPs (Table S4). It is noticeable that GO terms that have been previously identified by the enrichment analysis of the DE transcripts were also represented among enriched categories of SNP-harbouring transcripts, including those related to the antioxidant system and oxidationreduction processes (Table S3, Table S4, Fig. S2, Fig. S3). If these SNPs contribute to the differential expression of their respective transcripts remains elusive. 4. Discussion Human-caused radioactive contamination of the environment has a long-lasting impact on nature. As consequence of such events, natural populations are exposed to an extreme adverse condition for decades or hundreds of years. Unfortunately, these populations have no options other than adapt or face extinction. Given that plants are versatile organisms in terms of adaptation, much can be learnt by studying the strategies of these organisms in natura for overcoming chronic stress exposure. Scots pine is major species in boreal ecosystems and is widespread in the area affected by the Chernobyl NPP accident. Because of its high radiosensitivity (ICRP, 2008) and long lifespan, Scots pine is an interesting subject for

studying the biological consequences of chronic radiation exposure to biota species. With this background, we evaluated the transcriptome profile of pines belonging to populations under chronic radiation exposure to delineate the response pattern to this stress condition. The gene expression profile of the four studied populations correlated to the radiation levels to which they are exposed. The clustering analysis of the expression profiles grouped the two highly contaminated populations (Mas and Kul), while the low contaminated population (Zab) did not differ significantly from the reference (Fig. S1). Such a correlation is also supported by the high overlap of DE transcripts between Mas and Kul (Fig. 1). Nevertheless, Zab also shows a basal adaptive response as verified by the DE of stress-responsive transcripts and of those known to react to acute radiation exposure, all of which presenting the same expression pattern observed for Mas and Kul (Table 4, Table S3). It is interesting to note that of the seven transcripts which are commonly regulated among the three contaminated sample plots (Table 4), four are related to stress responses: a transcript for an anionic peroxidase related to cell death responses (WUN1_SOLTU), and three abscisic acid (ABA)-responsive transcripts (CIPKK_ARATH, CIPKA_ARATH, and SLAC1_ARATH). The F-box protein PP2-B11 (P2B11_ARATH), which was also down-regulated, has been described as an attenuator of ABA responses (Cheng et al., 2017). Nevertheless, the functional analysis of the DE transcripts does not evidence a classical stress signature response (e.g. heat, hypoxia, cold, etc.). In fact, the response to chronic radiation exposure also does not resemble one to acute radiation (Table S3), suggesting that the adaptation process to the former is rather unique. Those few genes that overlap between acute and chronic irradiation conditions showed inverse expression patterns (Table S3). Indeed, shortterm experiments have shown that the response pattern changes according to the radiation exposure duration (Kovalchuck et al., 2007). Considering that during an acute stress response the organism attempts to avoid damage even to the detriment of growth and development, as for instance starvation-induced autophagy or ABA-promoted growth arrest, it is plausible to consider that such a response pattern does not fit for chronic stress adaptation. For instance, it has been observed in feather grass from contaminated nuclear test areas in Kazakhstan the same levels of antioxidant enzymes than in plants from non-contaminated control region

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(Zaka et al., 2002). However, the former plants show an improved response to a new irradiation event than the latter, what has been attributed to the natural selection of the most adapted genotypes (Zaka et al., 2002). Although it could be the case for organisms with relatively short life cycle, recent studies suggest that a stress memory may play a role on the more efficient response during stress recurrence after an initial exposition (Crisp et al., 2016). Accordingly, evaluation of the antioxidant enzymes level of Scots pine populations from the radioactively contaminated Bryansk region, including Zab population, also did not show significant difference from the control (Volkova et al., 2017). Nevertheless, these plants are evidently influenced by radiation as revealed by the increased mutation rates (Volkova et al., 2017). The transcriptional profiles evaluated in our work further support this observation, as no transcripts encoding for antioxidant enzymes known to respond to acute radiation exposure were identified as DE. Conversely, our data suggests that the adaptation to chronic radiation exposure involves: (1) modulation of ROS accumulation, which must occur through the balance of ROS-generating processes and of antioxidant molecules; (2) control of cellular damage by enhanced expression of chaperones and histones, along with the modulation of ions balance, to counteract the damaging effects of a higher ROS basal level on proteins activity, DNA, and membranes; (3) control of the activity of TEs. The production of ROS is an inherent consequence of the exposure to radiation (Riley, 1994; Szumiel, 2015). During a ROS burst, plants activate antioxidant mechanisms which consist of enzymes (superoxide dismutase, catalases, peroxidases, and enzymes of ascorbate-glutathione cycle) and accumulation of low molecular weight antioxidants (ascorbic acid, glutathione, carotenoids, and atocopherol) for minimizing the damage (Sewelam et al., 2016). On the other hand, ROS are also central signalling molecules during stress responses (Sewelam et al., 2016). It is logical to hypothesize that organisms under chronic radiation exposure have to adapt to a different ROS threshold, which would allow them to balance the buffering of ROS without blocking their signalling function. Our data suggest that the ROS level modulation occurs mainly through the control of glutathione- and thioredoxin-related responses and most likely involving a fine-tuning of ROS-generating processes. Indeed, this result corroborates previous findings that in samples from Mas and Kul plots, the concentration of reduced glutathione is higher, while its oxidized form is present in lesser levels (Volkova et al., 2017). In this background of adaptation by modulation of ROS levels, the output given by the SNPs analysis is compelling, indicating enrichment of polymorphisms occurring on transcripts related to the antioxidant system and oxidation-reduction processes (Table S3, Table S4, Fig. S2, Fig. S3). Although most of the variations were identified in transcripts that are not DE, SNPs may confer stress toleration without influencing the transcription rate (Manishankar and Kudla, 2015). Indeed, local adaptation of different populations has been attributed to genetic variants (Namroud et al., 2008; Fournier-Level et al., 2011; Hancock et al., 2011). Although our study was based on somatic cells of needles, it would be interesting to evaluate if this pattern would also be observed on reproductive cells, especially of organisms with short life cycle. While processes that are sources of ROS are being repressed, several pieces of evidence suggest the occurrence of control of gas exchanges via stomata (Table 4; Table S3), what would directly impact on the photosynthesis rate, a major ROS source. It is interesting to note that the control of stomata movement, although relying on several ABA target genes, apparently does not involve the modulation of this hormone levels. Conversely, it rather occurs through the control of the sensitivity of the pathway via the repression of PP2-B11, a SnRK2 negative regulator (Table 4; Table S3). A perspective for future work is the confirmation of ABA

attenuation and of the control of photosynthesis rate on chronic irradiated plants. Interestingly, the transcriptome analysis revealed the absence of activation of repair processes in the populations under chronic radiation exposure, responses which are known to occur during acute radiation stress condition (Culligan et al., 2006). This observation can be understood as another evidence of the adaptation of the evaluated populations to the chronic stress, being the expression of the repair machinery below the detection threshold. Similarly, activation of the repair system was only identified in Lemna minor plants under high ionising radiation exposure (Van Hoeck et al., 2017). Nevertheless, the constant exposure to ionizing radiation imposes a permanent risk for the integrity of the molecules in the cells, including DNA single- and double-strand breaks (Caplin and Willey, 2018). The strategy adopted by the evaluated populations is likely the maintenance of the integrity of the molecules in the cells by the increased expression of chaperones and histones (Table S3). While HSPs have been reported to be induced by several stresses, including oxidative stress (Al-Whaibi, 2011), damage on DNA causes histones degradation (Hauer et al., 2017). Although the expression of histones is usually linked to the cell cycle (Jiang and Berger, 2017), it has been proposed that they have a radioprotective role (Oleinick et al., 1999), and that their synthesis is under a feedback control (Lee et al., 2012). In this context, the up-regulation of the transcription of histones would contribute to restore the histone supply necessary for the integrity of the DNA molecule. Another source of DNA damage which was found to be under control in the populations under chronic radiation exposure is the activity of TEs. The analysis of the activity of transposon families (Fig. 2) suggests that the regulation of such elements in response to chronic irradiation may be dose-dependent. While most of the TE families present induced activity in the low contaminated Zab plot, the opposite pattern is observed for the high contaminated Kul population, for which the TEs are mainly being repressed (Fig. 2). It is possible that the suppression of TEs under radiation influence may require a certain dose threshold, as it seems to occur for the control of gene expression observed for Zab population. Still, it is also feasible that the suppression of TEs activity in Kul plot is a consequence of a constitutively activated stress response mechanism. Indeed, our group has previously shown that genome hypermethylation is found on Scots pine trees from chronically irradiated areas (Volkova et al., 2018). Since a relation to the dose exposure was not evidenced, it is possible that the control of TEs activity in response to chronic radiation is not solely posttranscriptional (Volkova et al., 2018). In conclusion, the transcriptional profile of the response to chronic radiation exposure identified in our work will help to evaluate the impact and fate of populations facing such a long-term stress condition, and provides directions for future research. It is important to highlight that the unique stress profile shown by the three Scots pine populations evaluated is triggered by radiation doses lower than 130 mGy year1. Nonetheless, the radiation doses in the Chernobyl exclusion zone where the samples were collected are considered to be almost 30 times lower than the 400 mGy h1 (~3500 mGy year1) accepted as a safe for biota species (UNSCEAR, 2008). These doses are also lower than those recommended by the International Commission on Radiological Protection as safe for pine trees (4e40 mGy h1, ICRP, 2008). It is evident that the environmental management in radiation protection should be reviewed, while radiosensitive species should be used as models for such predictions. Data accessibility RNA-seq data is available in the Sequence Read Archive (https://

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