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Effect of modeled microgravity on radiation-induced adaptive response of root growth in Arabidopsis thaliana
Accepted Manuscript Title: Modulation of Modeled Microgravity on Radiation-Induced Adaptive Response of Root Growth in Arabidopsis thaliana Authors: Chenguang Deng, Ting Wang, Jingjing Wu, Wei Xu, Huasheng Li, Min Liu, Lijun Wu, Jinying Lu, Po Bian PII: DOI: Reference:
S0027-5107(16)30194-4 http://dx.doi.org/doi:10.1016/j.mrfmmm.2017.02.002 MUT 11579
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Mutation Research
Received date: Revised date: Accepted date:
13-12-2016 5-2-2017 10-2-2017
Please cite this article as: Chenguang Deng, Ting Wang, Jingjing Wu, Wei Xu, Huasheng Li, Min Liu, Lijun Wu, Jinying Lu, Po Bian, Modulation of Modeled Microgravity on Radiation-Induced Adaptive Response of Root Growth in Arabidopsis thaliana, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis http://dx.doi.org/10.1016/j.mrfmmm.2017.02.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Modulation of Modeled Microgravity on Radiation-Induced Adaptive Response of Root Growth in Arabidopsis thaliana Chenguang Deng
1, 2, 4
, Ting Wang1, 4, Jingjing Wu1, 2, Wei Xu1, Huasheng Li3, Min
Liu3, Lijun Wu1, Jinying Lu3*, Po Bian1, *
1. Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences; Key Laboratory of Environmental Toxicology and Pollution Control Technology of Anhui Province; Institute of Technical Biology and Agriculture Engineering; Chinese Academy of Sciences; 350 Shushanhu Road; Hefei 230031; P. R. China. 2. University of Science and Technology of China; Hefei 230026; P. R. China. 3. China Space Molecular Biological Lab, China Academy of Space Technology, Beijing 100086, P. R. China. 4. These authors contributed equally to this work. * Corresponding Author: Po Bian: P.O. Box 1138, Hefei, Anhui 230031, P. R. China. Tel: +86-551-65595145
Fax: +86-551-65595670
E-mail address: [email protected];
Jinying Lu: P.O. Box 2417-33, Beijing, 100081 P.R. China. Tel. :+86 1068379365 Fax:+86 1068379365 E-mail address: [email protected].
Highlights
The radio-adaptive response (RAR) of A. thaliana root growth is modulated in microgravity.
The DNA damage repairs in RAR are regulated by microgravity.
The phytohormone auxin plays a regulatory role in the modulation of microgravity on RAR of root growth.
Abstract Space particles have an inevitable impact on organisms during space missions; radio-adaptive response (RAR) is a critical radiation effect due to both low-dose background and sudden high-dose radiation exposure during solar storms. Although it is relevant to consider RAR within the context of microgravity, another major space environmental factor, to our knowledge, there is no evidence yet as to its effects on RAR. In the present study, we established an experimental method for detecting the effects of gamma-irradiation on the primary root growth of Arabidopsis thaliana, in which RAR of root growth was significantly induced by several dose combinations. Microgravity was simulated using a two-dimensional rotation clinostat. It was shown that RAR of root growth was significantly inhibited under the modeled microgravity condition, and was absent in pgm-1 plants that had impaired gravity sensing in root tips. These results suggest that RAR could be modulated in microgravity. Time course analysis showed that microgravity affected either the development of radio-resistance induced by priming irradiation, or the responses of plants to challenging irradiation.
Consistently, priming irradiation-induced expressions of DNA repair genes (AtKu70 and AtRAD54) were attenuated in microgravity, and reduced DNA repair efficiency in response to challenging irradiation was also observed after treatment with the modeled microgravity. In plant roots, the polar transportation of phytohormone auxin is regulated by gravity, and treatment with an exogenous auxin (indole-3-acetic acid) prevented the induction of RAR of root growth, suggesting that auxin might play a regulatory role in the interaction between microgravity and RAR of root growth.
Keywords: modeled microgravity; radio-adaptive response; Arabidopsis thaliana; root growth; DNA repair pathway; auxin
1. Introduction During space flights, astronauts and other living organisms are inevitably exposed to space radiation composed of protons and electrons captured by the Earth’s magnetic field (Van Allen belts), and protons and high atomic number particles from continuous galactic cosmic rays (GCR) and sudden solar particle events. Moreover, inside the spacecraft, there are secondary ionization events (neutrons and recoil nuclei) excited by the interaction of space particles with spacecraft shielding and internal objects [1, 2]. One of the characteristics of space radiation is the chronic exposure at a low dose and low fluence rate. It has been estimated that inside the Russian Space Station “Mir”, a single lymphocyte in the body of an astronaut is traversed by one proton every 12 days, one helium ion in 4 months, one oxygen ion in 24 years, and one iron ion in 400 years [3]. Thus low-dose and low-fluence space radiation gives rise to two critical radiation biological effects: radiation-induced bystander effect (RIBE) and radio-adaptive response (RAR) [4–6]. Another characteristic of space radiation is temporal and spatial heterogeneity. When astronauts and other living organisms in spacecraft travel from a low-dose region to a high-dose region, or encounter sudden exposure to high-dose radiation from solar storms, the RAR initiated by low-dose radiation attenuates the detrimental consequences caused by the
high-dose radiation. For instance, in deep space missions, chronic exposure to elevated levels of GCR radiation can greatly decrease radiation susceptibility and better protect astronauts against unpredictable exposures to sudden and dramatic increases in flux due to solar flares and coronal mass ejections [6]. Therefore, RAR is thought to be an essential factor for estimating potential health risks for astronauts, and is a critical consideration for biological protection against space radiation [6]. It has been proposed that for long-term space travel, astronauts or organisms with high adaptive response should be chosen [6]. In addition to space radiation, microgravity (10-4-10-6g) is another major space environmental factor. Microgravity can cause declines in cellular immune function [7, 8], cardiovascular changes [9], bone loss [10], muscular atrophy [11], and renal stones [12] in humans and mammals. For higher plants, development, growth, metabolism, and signaling transduction are changed to different extents under microgravity conditions [13]. Interestingly, the biological effects of ionizing radiation could also be modified by microgravity through additive, synergistic, or antagonistic modalities [14]. Recently, we reported the modulation of modeled microgravity on the RIBE in the model plant Arabidopsis thaliana (A. thaliana), where some biological processes, such as RIBE-meditated expressions of AtRAD54 and AtRAD51 genes and alleviation of TGS-silenced locus, are significantly inhibited in the modeled microgravity [15]. Considering the mechanistic links between RIBE and RAR [16–18], it was proposed that RAR should also be affected by microgravity. However, there is no evidence regarding the effect of microgravity on the RAR. Plants have evolved adaptive responses to cope with a variety of biotic and abiotic environmental stress at the physiological, genetic, and epigenetic levels [19, 20]. Adaptive response is demonstrated in the root cells of Allium cepa when exposed to low aluminum concentrations and subsequent genotoxic chemical agents such as ethyl methane sulfonate [21]. The plant root is a key organ in the response to gravity, in which the gravity is sensed through sediment of starch statoliths in the columella cells in the root cap [22]. In our previous study, it has been shown that A. thaliana root growth is significantly inhibited by ionizing irradiation through RIBE [23, 24]. Thus,
in this study, we first established an experimental model of RAR with root growth as the biological endpoint, and then demonstrated the manifestation of RAR of root growth in microgravity using a two-dimensional rotation clinostat.
2. Materials and methods 2.1 A. thaliana lines and plant growth The A. thaliana wild-type (Col-0) and mutant line pgm-1 were obtained from the NASC (Nottingham Arabidopsis Stock Center, UK). The pgm-1 plant is unable to synthesize starch due to lack of the gene encoding the plastidic phosphoglucomutase (PGM) [25]. The transgenic line DR5 was a kind gift from Prof. Guifoyle (Department of Biochemistry, University of Missouri), in which the DR5-GUS construct contained seven copies of DR5 (an auxin-responsive TGTCTC element) cloned upstream of the minimal 46 cauliflower mosaic virus (CaMV) 35S promoter-GUS reporter [26]. Surface-sterilized seeds of A. thaliana were sown on growth medium (1× Murashige and Skoog (MS) mineral salts, agar at 0.8% (w/v), and sucrose at 1% (w/v)) in Petri dishes. After 48 h of stratification at 4°C, the Petri dishes were placed in a growth chamber at 22°C with a continuous illumination of approximately 100 μMm2 s-1 in a vertical orientation so that the roots would grow along the agar surface. 2.2 Microgravity simulation and gamma-irradiation The microgravity effect of A. thaliana seedlings was simulated using a two-dimensional rotation clinostat, made by the Center for Space Science and Applied Research of the Chinese Academy of Sciences. The rotation clinostat was horizontally placed, with the rotating discs in a vertical orientation. The Petri dishes with seedlings were fixed on the rotating disk, with the root tips of seedlings toward the rotating center, as shown in Figure 1. The rotation clinostat rotated continuously at a speed of 2 rpm, and the distance from root tips to the rotating center measured approximately 12 cm. In this condition, the seedlings were subjected to a modeled microgravity of approximately 5×10-4g. Four-day-old seedlings were treated with 2–150 Gy of gamma-irradiation at a
dose rate of 3.37 Gy/min using a Biobeam Cs137 irradiator (cat no. GM 2000; Gamma-Service Medical, Leipzig, Germany). In RAR experiments, the seedlings were first irradiated with low priming doses (2–20 Gy), and then placed in the rotation clinostat for given durations under culture conditions similar to the growth chamber. After the microgravity treatment, seedlings were again gamma-irradiated with high challenging doses (25–150 Gy), and then grow in the growth chamber until detected, as shown in Figure 1. 2.3 Measurement of primary root growth of A. thaliana The elongation of primary roots of seedlings was measured using Image J software (National Institutes of Health; http://rsb.info.nih.gov/ij) from digital images captured with a Nikon D600 camera. The final data were the average of three independent experiments, and 15-20 seedlings were scored for each experiment. 2.4 qRT- PCR analysis of the expressions of the AtRAD54 and AtKu70 genes In each experiment, 15-20 roots were sampled and homogenized in liquid nitrogen. Total mRNAs were extracted using Trizol reagent (Invitrogen, USA) according to manufacturer’s protocols. Total RNA (5 µg) was reverse-transcribed at 42°C for 1 h using Transcript one-step gDNA removal and cDNA synthesis supermix kit (Transgen Biotech, China) according to manufacturer’s protocols. The reaction mixture of qRT-PCR (total 20 µl) contained 2µl of cDNA as a template, 50 nM primers, 10 µl of 2× SYBR Premix Ex Taq (Takara, Japan), and 0.4 µl of 50 × ROX Reference Dye. Primers for the AtUBQ5, AtRAD54, and AtKu70 genes were used according to previous study [27]. The mRNA levels of the AtRAD54 and AtKu70 genes were measured using the Applied Biosystems StepOneTM qRT-PCR device (Life technologies, Singapore) under the following conditions: one cycle of 95°C for 10 s, and then 40 cycles of 95°C for 5 s and 60°C for 31 s. The AtUBQ5 gene was used as an internal control. The final data were the average of 3 independent experiments, with 3 technical replicates for each experiment. 2.5 Treatment of seedlings with exogenous indole-3-acetic acid (IAA) IAA (Sigma Aldrich, St Louis, MO, USA) was dissolved in 100% ethanol, and the stock solution was stored at 4°C. IAA was added into growth medium at a
concentration of 1 nM [28], with less than 0.01% of ethanol (v/v) in the growth medium. Control treatments were applied using equal amounts of ethanol solvent without IAA. After priming irradiation, seedlings were transferred onto the IAA medium for 8 h, and continued to grow for 6 days on IAA medium after challenging irradiation. 2.6 Quantitative analysis and histochemical staining of GUS activity After treatments with gamma-irradiation and/or microgravity, 5-mm long root distal parts were sampled, and quantitative GUS activity was assayed according to protocols described previously [29, 30]. The fluorescence at 455 nm under excitation at 365 nm was measured with a luminescence spectrophotometer equipped with an ELISA plate reader (Spectra Max M2, Molecular Devices, USA). The final data were the average of 15 samples from three independent experiments. For detection of GUS distribution in root tips, the roots of line DR5 were collected after treatments with gamma-irradiation and/or microgravity, and then stained as described previously [31]. 2.7 Statistical Analysis All results are presented as means ± standard deviations. The statistical significance of the experiments was determined by performing Student’s t test. A P value of 0.05 or less was considered significant.
3. Results 3.1 Root growth RAR of A. thaliana
It is well known that one way that plants sense gravity is via physical sedimentation of the statoliths in columella cells in root tips [22]. Thus, in order to investigate the interaction between microgravity and RAR, an experimental model of RAR with plant roots as a checking object was first required. It has been reported that plant root growth is sensitive to ionizing radiation [23, 24]. Therefore, we first tested the relationship between irradiation dose and root growth using 4-day-old seedlings. Low doses of gamma-irradiations (2, 5, 10, and 20 Gy) did not affect the growth of primary roots during various periods after irradiation (in all cases, P > 0.05), as shown in Figure 2A. High gamma-radiation doses (25, 50, 75, 100, 125, and 150 Gy) significantly repressed root growth in a dose-dependent manner (in all cases, P < 0.01), as shown in Figure 2B. It should be noted that root growth within 1 day after irradiation was unchanged (in all cases, P > 0.05), but root growth was suppressed within 2-3 days (in all cases, P < 0.01), and more remarkably within 2-6 days (in all cases, P < 0.01) after irradiation (Figure 2B). Therefore, in the following experiments, root growth within 2-6 days after irradiation was scored, unless otherwise specified. The dose combinations for RAR were further tested. The plants were respectively irradiated with 2, 5, 10, and 20 Gy of gamma-rays (priming doses), and then subjected to 100 Gy of gamma-rays (challenging dose) at an 8 h interval. As shown in Figure 3A, only 10 Gy of priming irradiation significantly alleviated the repressive effect of challenging irradiation on root growth (P < 0.01). In the following experiments, 10 Gy of gamma-irradiation was used as priming dose, and 25, 50, 75, 100, 125, and 150 Gy of gamma-irradiations were used as challenging doses. The RAR was significantly induced by the 10+25 Gy, 10+50 Gy, 10+75 Gy, 10+100 Gy, and 10+125 Gy dose combinations (in all cases, P < 0.05), as shown in Figure 3B. The other time intervals between priming and challenging irradiations (2, 4, 8, 12, 16, and 24 h) were also tested using the 10+100 Gy dose combination. It was shown that the RAR could be induced with time intervals of 4, 8, and 12 h (in all cases, P < 0.01), as shown in Figure 3C. Therefore, in the following experiments, the 10+100 Gy dose combination and 8-h interval were used, unless otherwise specified. 3.2 Effect of modeled microgravity on RAR of root growth
Based on the established experimental model for RAR of root growth, the effect of microgravity on the RAR was further investigated through applying microgravity within the 8 h interval between the priming and challenging irradiations. As shown in Figure 4A, microgravity itself did not interrupt the root growth of naïve plants and plants subjected to 10 and 100 Gy of gamma-irradiations (in all cases, P > 0.05). However, the application of the modeled microgravity significantly prevented the induction of RAR by 10+100 Gy (P > 0.05), as shown in Figure 4B. Moreover, the induction of RAR by the dose combinations of 10+50 Gy and 10+75 Gy was suppressed by the modeled microgravity (in both cases, P > 0.05), as shown in Figure 4C. These results suggest that root growth RAR could be modulated in the modeled microgravity. In order to further strengthen this conclusion, the A. thaliana pgm-1 plants, which are impaired in root gravity sensing due to lack of starch statoliths in the columella cells in the root cap, were also tested. Under normal gravity conditions, the mutant plants also exhibited a suppressed induction of RAR by the10+100 Gy dose combination (P > 0.05), but not by the 10+50 Gy and 10+75 Gy dose combinations (in both cases, P < 0.01), as shown in Figure 4D. 3.3. Time course of interaction between modeled microgravity and RAR In RAR, radio-resistance is developed between priming and challenging irradiations. Here, we investigated the effect of microgravity on the development of RAR. For this purpose, the plants were subjected to microgravity for 0, 2, 4, 6, and 8 h after priming irradiation, respectively, and then exposed to challenging irradiation at 8 h after priming irradiation. The results showed that the induction of RAR was prevented by the application of microgravity within 0-6 and 0-8 h after priming irradiation (in both cases, P > 0.05), but not within 0-0, 0-2, and 0-4 h after priming irradiation (in all cases, P < 0.01), as shown in Figure 5A. Moreover, seedlings were placed in the modeled microgravity at time points of 0, 2, 4, 6, and 8 h after priming irradiation until the application of challenging irradiation, respectively. As shown in Figure 5B, the induction of RAR was significantly suppressed by the application of the modeled microgravity within 0-8 and 2-8 h after priming irradiation (in both cases,
P > 0.05), but not within 4-8, 6-8, and 8-8 h (in all cases, P < 0.01). These results suggest that 6-h duration of microgravity might be required to suppress the development of radio-resistance. The seedlings were also placed in the modeled microgravity for 8 h before priming irradiation. Plant memory of microgravity did not affect the induction of RAR (P < 0.01), as shown in Figure 5C. In another experiment, the plants were subjected to the modeled microgravity for 8 h after treatment with challenging irradiation. Interestingly, the RAR could be suppressed by the later application of microgravity (P > 0.05), as shown in Figure 5D. 3.4 Role of DNA damage repair in RAR of root growth It has been reported that DNA damage repairs play a pivotal role in the induction of RAR [32–34]. In A. thaliana, the non-homologous end joining (NHEJ) and homologous recombination (HR) are two important DNA repair machineries [35], in which the AtRAD54 (HR) and AtKu70 (NHEJ) genes are transcriptionally inducible in response to DNA-damaging agents [36–38]. In order to further know whether microgravity affects the repairing of DNA damage caused by challenging irradiation in root cells, the expressions of the AtKu70 and AtRAD54 genes were detected using qRT-PCR. At 8 h after priming irradiation, just before the application of challenging irradiation, the 10 Gy of gamma-irradiation led to up-regulated expressions of these genes (in both cases, P < 0.01). Interestingly, priming irradiation-induced expressions of AtKu70 and AtRAD54 genes were dramatically down-regulated in the modeled microgravity compared to the 10 Gy irradiation alone (in both cases, P < 0.01) (Figure 6A), suggesting that microgravity might change the priming of DNA damage repairs induced by priming irradiation. The challenging irradiation alone (100 Gy) also led to significantly up-regulated expressions of the AtKu70 and AtRAD54 genes at 2, 12, and 24 h after challenging irradiation (in all cases, P < 0.01), as shown in Figure 6B and C. In normal gravity, their expressions were significantly down-regulated at 12 and 24 h (in all cases, P < 0.01), but not at 2 h (in both cases, P > 0.05) in the irradiation with dose combination of 10+100 Gy compared to the 100 Gy of gamma-irradiation alone, as shown in Figure 6B and C, suggesting that the priming irradiation might promote
the efficiency of DNA damage repair in root cells in response to the challenging irradiation. However, in microgravity, the expression of the AtKu70 gene induced by 10+100 Gy was not down-regulated at 12 h after challenging irradiation compared to the 100 Gy of gamma-irradiation alone (P > 0.05) (Figure 6B), suggesting reduced efficiency of DNA damage repairs in microgravity. 3.5 Role of auxin in modulation of RAR in modeled microgravity It is well accepted that the phytohormone auxin is a key regulator of cell division and elongation [39]. Its polar transport in root tips is regulated by the sedimentation of the statoliths in columella cells [22]. Thus, a question arises as to whether auxin is involved in the interaction between microgravity and RAR of root growth. To address this, seedlings were transferred onto medium containing 1nM of IAA after receiving priming irradiation. The treatment with IAA itself did not affect the root growth of naïve plants or plants irradiated with 10 or 100 Gy of gamma-rays (in all cases, P > 0.05). Interestingly, the induction of RAR of root growth was significantly suppressed in the presence of the IAA (P > 0.05), as shown in Figure 7A. Consistently, the up-regulated expressions of the AtKu70 and AtRAD54 genes by priming irradiation were also significantly suppressed in the presence of IAA (in both cases, P < 0.01), as shown in Figure 7B. The level of auxin in root tips was also measured using DR5 plants, in which GUS activity correlates positively with the auxin level. The auxin level in root tips was not changed by the 10 Gy of priming irradiation (Figure 7C), or 100 Gy of challenging irradiation alone (Figure 7D) (in both cases, P > 0.05). However, irradiation with the 10+100 Gy dose combination led to a decreased level of auxin in the root tips (P < 0.05) (Figure 7D). Interestingly, although the auxin level was not affected in microgravity at 8 h after priming irradiation (P > 0.05) (Figure 7C), the decreased auxin level induced by the 10+100 Gy dose combination was eliminated under the modeled microgravity condition (P > 0.05), as shown in Figure 7D. Similarly, histochemical staining showed an enlarged distribution of auxin in root tips subjected to the 10+100 Gy dose combination in microgravity compared to that in normal gravity, as shown in Figure 7E.
Discussion Space is a complex environment, and many factors, such as radiation particles and microgravity, affect each other at various biological levels [13]. Therefore, their biological effects should be considered in the presence of other factors. Due to the temporal and spatial heterogeneity of space radiation, the RAR is thought to have a potential effect on space crews and the other living organisms carried by spacecraft. Here we present the first evidence that the RAR can be modulated in microgravity. However, the effect of microgravity on RAR was demonstrated only using the root growth of A. thaliana as the biological endpoint; it is unclear whether the RAR of other biological events in different organs or whole organisms, if any, is also modulated by microgravity in the same way. Moreover, in this study the RAR of root growth of A. thaliana was induced by acute exposure to gamma-rays (3.37 Gy/min), and also with higher doses of priming irradiation (10 Gy) and challenging irradiations (25–125 Gy) due to the high radio-resistance of higher plants [40]. Thus, the irradiation pattern and doses used here are not very relevant to a space environment characterized by low-dose and low-fluence radiation. Therefore, in this study, the modulation of RAR by microgravity was demonstrated only in principle, and highly radiosensitive experimental models might be required to demonstrate this phenomenon under more relevant radiation patterns and doses. For higher plants, root growth is related to two zones in the root tip: the meristematic zone (MZ) and the elongation zone (EZ). The root apical meristem (RAM) in the MZ provides new cells for the growing root, and the expansion of root cells in the EZ contributes to primary root growth [39]. In this study, root growth was not affected within 1 day after various doses of gamma-irradiation (Figure 2A), but was significantly suppressed within 2-3 and 2-6 days after irradiation (Figure 2B). It has also been reported that dividing cells are more sensitive to radiation than differentiated cells [41]. Therefore, the repressive effect of gamma-irradiation on root growth might be mainly due to the decreased division of RAM cells. Thus, the
priming irradiation might only up-regulate the radio-resistance of RAM cells, at least in terms of the induction of RAR of root growth. Moreover, it was shown that the radio-resistance induced by priming irradiation persisted only for 12 hours (Figure 3C), whereas the effect of RAR on root growth lasted for 2-6 days after challenging irradiation, during which the RAM has undergone multiple generations of cell divisions. If radio-resistance is induced only in these dividing RAM cells, the effect of RAR on root growth will disappear shortly after these cells enter the EZ. In addition to the dividing cells, the RAM also contains a set of initial cells (stem cells) [39]. The challenging irradiation alone suppressed the root growth within 2–6 days after irradiation (Figure 2B), suggesting that resulting DNA damage in RAM stem cells may persist for a longer time, also possibly transmitted into their progeny cells. Thus, it is likely that the priming irradiation might initiate the radio-resistance in the RAM stem cells that reduces the challenging irradiation-induced persistent DNA damage in these cells. This might be a reasonable explanation for the long-term effect of RAR on root growth. On the other hand, the modeled microgravity inhibited the RAR induced by dose combinations of 10+50 Gy, 10+75 Gy, and 10+100 Gy (see Figure 4B and C), whereas the deficiency of gravity sensation in pgm-1 plants only prevented the induction of RAR by the 10+100 Gy dose combination (see Figure 4D). Like pgm-1 line, the A. thaliana TC7 mutant has been reported to be starch-free and still exhibit root gravitropism [42], indicating that, in addition to the starch-statolith machinery, higher plants also employ other gravity-sensing machinery such as the gravitational pressure model proposed by the Cholodny–Went hypothesis [43]. It is therefore likely that in pgm-1 plants, residual gravity sensing in other cells might attenuate the repressive effect of the absence of starch-statolith machinery on the induction RAR of root growth compared to the whole-body absence of gravity under the modeled microgravity condition. In the present study, treatment with exogenous IAA was shown to inhibit the induction of RAR of root growth (Figure 7A). Moreover, 10 Gy of priming irradiation or 100 Gy of challenging irradiation alone did not affect the level and distribution of auxin in root tips (Figure 7C, D, and E); however, the plants subjected to the 10+100
Gy dose combination exhibited a decreased level of auxin in root tips at 12 h after challenging irradiation (Figure 7D and E). These results indicate that auxin might play a regulatory role in the induction of RAR of root growth. Auxin is one of key regulators of cell division in the MZ of roots, and the genes in auxin biosynthesis are also expressed specifically in the root stem cell niche [44–46]. Disturbances in auxin level and distribution can change the cycle phases of RAM cells [39], to which the radio-sensitivity of cells is closely related. It has been reported that the induction of RAR is largely dependent on radio-sensitivity [32]. Moreover, DNA damage repairs also play a critical role in the induction of RAR [32–34]. It has been reported that the expression of the Ku gene in hypocotyls of Vigna radiate is regulated by auxin [47]. In this study, the priming irradiation-induced expressions of the AtKu70 and AtRAD54 genes were suppressed in the presence of IAA (Figure 7B). Therefore, it is likely that auxin might affect the induction of RAR of root growth through changing the radio-sensitivity and/or DNA repair of RAM cells. In plant root tips, polar transportation of auxin is regulated by gravity via the starch-statolith machinery [22]. For the 10+100 Gy dose combination, a different level and distribution of auxin in the root tips was also observed at 12 h after challenging irradiation in microgravity compared to that in normal gravity (Figure 7D and E). Therefore we reason that microgravity suppresses the induction of RAR of root growth through interfering with the level and distribution of auxin in root tips. In addition to auxin, other plant hormones such as abscisic acid (ABA) and gibberellic acid (GA) also take part in the regulation of cell division in the MZ [39] and in DNA damage repairs [48, 27]. However, due to the absence of evidence about the regulations of microgravity on their transportation and signaling transduction, it does not appear that they play regulatory roles in interaction between microgravity and RAR of root growth. However, it is likely that microgravity could regulate them through cross-talk of signaling between auxin and these plant hormones [49, 50].
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments We thank Prof. Guifoyle and NASC for their generous provision of the various A. thaliana seeds. This work was supported by the National Science Fund of China (11575233, 11275230), Natural Science Fund of Anhui Province (1408085MKL51) and International S&T Cooperation Program of China (2015DFR30920).
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Legends Fig.1 Schematic representation of gamma-irradiation and microgravity simulation of Arabidopsis thaliana.
Fig.2 The dose relationship of root growth of A. thaliana seedlings. A) Root growth at indicated periods after 2–20 Gy of gamma-irradiation; B) Root growth at indicated periods after 25–150 Gy of gamma-irradiation. Results are means ± SD (n = 3, t test, ** P < 0.01).
Fig.3 Induction of RAR of root growth by combined gamma-irradiations. A) Root growth of seedlings subjected to 2, 5, 10, and 20 Gy of priming gamma-irradiations, and 100 Gy of challenging gamma-irradiation; B) Root growth of seedlings subjected to 10 Gy of priming gamma-irradiation and 25, 50, 75, 100, 125, and 150 Gy of challenging gamma-irradiations; C) Effect of time intervals between priming and challenging irradiations on the induction of RAR. Results are means ± SD (n = 3, t test, * P < 0.05, ** P < 0.01).
Fig.4 Modulation of microgravity on the RAR of root growth. A) Effect of microgravity on the root growth of naïve or irradiated seedlings; B) Effect of modeled microgravity on the RAR induced by 10+100 Gy dose combination; C) Effect of modeled microgravity on the RAR induced by 10+50 Gy and 10+75 Gy dose combinations; D) The induction of RAR in pgm-1 plants deficient in root gravity sensing. Results are means ± SD (n = 3, t test, ** P < 0.01).
Fig.5 Time course of interaction between RAR and microgravity. A) The induction of RAR in seedlings placed in modeled microgravity for the indicated durations after priming irradiation; B) The induction of RAR in seedlings placed in modeled microgravity at time points of 0, 2, 4, 6, and 8 h after priming irradiation until 8 h after priming irradiation; C) The induction of RAR in seedlings placed in modeled
microgravity for 8 h before priming irradiation; D) The induction of RAR in seedlings placed in modeled microgravity for 8 h after challenging irradiation. Results are means ± SD (n = 3, t test, ** P < 0.01).
Fig.6 Expressions of DNA repair genes in roots after treatments with gamma-irradiation and/or modeled microgravity. A) Effect of microgravity on mRNA levels of the AtKu70 and AtRAD54 genes at 8 h after priming irradiation; B) mRNA level of the AtKu70 gene at 2, 12, and 24 h after challenging irradiation under the normal gravity (NG) and modeled microgravity (MG); C) mRNA level of the AtRAD54 gene at 2, 12, and 24 h after challenging irradiation in the normal gravity (NG) and modeled microgravity (MG). Results are means ± SD (n = 3, t test, ** P < 0.01).
Fig.7 Effect of the phytohormone auxin on the induction of RAR. A) Effect of exogenous IAA on the induction of RAR of root growth; B) mRNA levels of the AtKu70 and AtRAD54 genes at 8 h after priming irradiation in the presence of IAA; C) Level of auxin in root tips at 8 h after priming irradiation; D) Level of auxin in root tips at 12 h after challenging irradiation; E) Histochemical staining of GUS activity in DR5 seedlings at 12 h after challenging irradiation. Results are means ± SD (n = 3 for analysis of root growth, n = 15 for analysis of GUS activity, t test, * P < 0.05, ** P < 0.01).
S0027-5107(16)30194-4 http://dx.doi.org/doi:10.1016/j.mrfmmm.2017.02.002 MUT 11579
To appear in:
Mutation Research
Received date: Revised date: Accepted date:
13-12-2016 5-2-2017 10-2-2017
Please cite this article as: Chenguang Deng, Ting Wang, Jingjing Wu, Wei Xu, Huasheng Li, Min Liu, Lijun Wu, Jinying Lu, Po Bian, Modulation of Modeled Microgravity on Radiation-Induced Adaptive Response of Root Growth in Arabidopsis thaliana, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis http://dx.doi.org/10.1016/j.mrfmmm.2017.02.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Modulation of Modeled Microgravity on Radiation-Induced Adaptive Response of Root Growth in Arabidopsis thaliana Chenguang Deng
1, 2, 4
, Ting Wang1, 4, Jingjing Wu1, 2, Wei Xu1, Huasheng Li3, Min
Liu3, Lijun Wu1, Jinying Lu3*, Po Bian1, *
1. Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences; Key Laboratory of Environmental Toxicology and Pollution Control Technology of Anhui Province; Institute of Technical Biology and Agriculture Engineering; Chinese Academy of Sciences; 350 Shushanhu Road; Hefei 230031; P. R. China. 2. University of Science and Technology of China; Hefei 230026; P. R. China. 3. China Space Molecular Biological Lab, China Academy of Space Technology, Beijing 100086, P. R. China. 4. These authors contributed equally to this work. * Corresponding Author: Po Bian: P.O. Box 1138, Hefei, Anhui 230031, P. R. China. Tel: +86-551-65595145
Fax: +86-551-65595670
E-mail address: [email protected];
Jinying Lu: P.O. Box 2417-33, Beijing, 100081 P.R. China. Tel. :+86 1068379365 Fax:+86 1068379365 E-mail address: [email protected].
Highlights
The radio-adaptive response (RAR) of A. thaliana root growth is modulated in microgravity.
The DNA damage repairs in RAR are regulated by microgravity.
The phytohormone auxin plays a regulatory role in the modulation of microgravity on RAR of root growth.
Abstract Space particles have an inevitable impact on organisms during space missions; radio-adaptive response (RAR) is a critical radiation effect due to both low-dose background and sudden high-dose radiation exposure during solar storms. Although it is relevant to consider RAR within the context of microgravity, another major space environmental factor, to our knowledge, there is no evidence yet as to its effects on RAR. In the present study, we established an experimental method for detecting the effects of gamma-irradiation on the primary root growth of Arabidopsis thaliana, in which RAR of root growth was significantly induced by several dose combinations. Microgravity was simulated using a two-dimensional rotation clinostat. It was shown that RAR of root growth was significantly inhibited under the modeled microgravity condition, and was absent in pgm-1 plants that had impaired gravity sensing in root tips. These results suggest that RAR could be modulated in microgravity. Time course analysis showed that microgravity affected either the development of radio-resistance induced by priming irradiation, or the responses of plants to challenging irradiation.
Consistently, priming irradiation-induced expressions of DNA repair genes (AtKu70 and AtRAD54) were attenuated in microgravity, and reduced DNA repair efficiency in response to challenging irradiation was also observed after treatment with the modeled microgravity. In plant roots, the polar transportation of phytohormone auxin is regulated by gravity, and treatment with an exogenous auxin (indole-3-acetic acid) prevented the induction of RAR of root growth, suggesting that auxin might play a regulatory role in the interaction between microgravity and RAR of root growth.
Keywords: modeled microgravity; radio-adaptive response; Arabidopsis thaliana; root growth; DNA repair pathway; auxin
1. Introduction During space flights, astronauts and other living organisms are inevitably exposed to space radiation composed of protons and electrons captured by the Earth’s magnetic field (Van Allen belts), and protons and high atomic number particles from continuous galactic cosmic rays (GCR) and sudden solar particle events. Moreover, inside the spacecraft, there are secondary ionization events (neutrons and recoil nuclei) excited by the interaction of space particles with spacecraft shielding and internal objects [1, 2]. One of the characteristics of space radiation is the chronic exposure at a low dose and low fluence rate. It has been estimated that inside the Russian Space Station “Mir”, a single lymphocyte in the body of an astronaut is traversed by one proton every 12 days, one helium ion in 4 months, one oxygen ion in 24 years, and one iron ion in 400 years [3]. Thus low-dose and low-fluence space radiation gives rise to two critical radiation biological effects: radiation-induced bystander effect (RIBE) and radio-adaptive response (RAR) [4–6]. Another characteristic of space radiation is temporal and spatial heterogeneity. When astronauts and other living organisms in spacecraft travel from a low-dose region to a high-dose region, or encounter sudden exposure to high-dose radiation from solar storms, the RAR initiated by low-dose radiation attenuates the detrimental consequences caused by the
high-dose radiation. For instance, in deep space missions, chronic exposure to elevated levels of GCR radiation can greatly decrease radiation susceptibility and better protect astronauts against unpredictable exposures to sudden and dramatic increases in flux due to solar flares and coronal mass ejections [6]. Therefore, RAR is thought to be an essential factor for estimating potential health risks for astronauts, and is a critical consideration for biological protection against space radiation [6]. It has been proposed that for long-term space travel, astronauts or organisms with high adaptive response should be chosen [6]. In addition to space radiation, microgravity (10-4-10-6g) is another major space environmental factor. Microgravity can cause declines in cellular immune function [7, 8], cardiovascular changes [9], bone loss [10], muscular atrophy [11], and renal stones [12] in humans and mammals. For higher plants, development, growth, metabolism, and signaling transduction are changed to different extents under microgravity conditions [13]. Interestingly, the biological effects of ionizing radiation could also be modified by microgravity through additive, synergistic, or antagonistic modalities [14]. Recently, we reported the modulation of modeled microgravity on the RIBE in the model plant Arabidopsis thaliana (A. thaliana), where some biological processes, such as RIBE-meditated expressions of AtRAD54 and AtRAD51 genes and alleviation of TGS-silenced locus, are significantly inhibited in the modeled microgravity [15]. Considering the mechanistic links between RIBE and RAR [16–18], it was proposed that RAR should also be affected by microgravity. However, there is no evidence regarding the effect of microgravity on the RAR. Plants have evolved adaptive responses to cope with a variety of biotic and abiotic environmental stress at the physiological, genetic, and epigenetic levels [19, 20]. Adaptive response is demonstrated in the root cells of Allium cepa when exposed to low aluminum concentrations and subsequent genotoxic chemical agents such as ethyl methane sulfonate [21]. The plant root is a key organ in the response to gravity, in which the gravity is sensed through sediment of starch statoliths in the columella cells in the root cap [22]. In our previous study, it has been shown that A. thaliana root growth is significantly inhibited by ionizing irradiation through RIBE [23, 24]. Thus,
in this study, we first established an experimental model of RAR with root growth as the biological endpoint, and then demonstrated the manifestation of RAR of root growth in microgravity using a two-dimensional rotation clinostat.
2. Materials and methods 2.1 A. thaliana lines and plant growth The A. thaliana wild-type (Col-0) and mutant line pgm-1 were obtained from the NASC (Nottingham Arabidopsis Stock Center, UK). The pgm-1 plant is unable to synthesize starch due to lack of the gene encoding the plastidic phosphoglucomutase (PGM) [25]. The transgenic line DR5 was a kind gift from Prof. Guifoyle (Department of Biochemistry, University of Missouri), in which the DR5-GUS construct contained seven copies of DR5 (an auxin-responsive TGTCTC element) cloned upstream of the minimal 46 cauliflower mosaic virus (CaMV) 35S promoter-GUS reporter [26]. Surface-sterilized seeds of A. thaliana were sown on growth medium (1× Murashige and Skoog (MS) mineral salts, agar at 0.8% (w/v), and sucrose at 1% (w/v)) in Petri dishes. After 48 h of stratification at 4°C, the Petri dishes were placed in a growth chamber at 22°C with a continuous illumination of approximately 100 μMm2 s-1 in a vertical orientation so that the roots would grow along the agar surface. 2.2 Microgravity simulation and gamma-irradiation The microgravity effect of A. thaliana seedlings was simulated using a two-dimensional rotation clinostat, made by the Center for Space Science and Applied Research of the Chinese Academy of Sciences. The rotation clinostat was horizontally placed, with the rotating discs in a vertical orientation. The Petri dishes with seedlings were fixed on the rotating disk, with the root tips of seedlings toward the rotating center, as shown in Figure 1. The rotation clinostat rotated continuously at a speed of 2 rpm, and the distance from root tips to the rotating center measured approximately 12 cm. In this condition, the seedlings were subjected to a modeled microgravity of approximately 5×10-4g. Four-day-old seedlings were treated with 2–150 Gy of gamma-irradiation at a
dose rate of 3.37 Gy/min using a Biobeam Cs137 irradiator (cat no. GM 2000; Gamma-Service Medical, Leipzig, Germany). In RAR experiments, the seedlings were first irradiated with low priming doses (2–20 Gy), and then placed in the rotation clinostat for given durations under culture conditions similar to the growth chamber. After the microgravity treatment, seedlings were again gamma-irradiated with high challenging doses (25–150 Gy), and then grow in the growth chamber until detected, as shown in Figure 1. 2.3 Measurement of primary root growth of A. thaliana The elongation of primary roots of seedlings was measured using Image J software (National Institutes of Health; http://rsb.info.nih.gov/ij) from digital images captured with a Nikon D600 camera. The final data were the average of three independent experiments, and 15-20 seedlings were scored for each experiment. 2.4 qRT- PCR analysis of the expressions of the AtRAD54 and AtKu70 genes In each experiment, 15-20 roots were sampled and homogenized in liquid nitrogen. Total mRNAs were extracted using Trizol reagent (Invitrogen, USA) according to manufacturer’s protocols. Total RNA (5 µg) was reverse-transcribed at 42°C for 1 h using Transcript one-step gDNA removal and cDNA synthesis supermix kit (Transgen Biotech, China) according to manufacturer’s protocols. The reaction mixture of qRT-PCR (total 20 µl) contained 2µl of cDNA as a template, 50 nM primers, 10 µl of 2× SYBR Premix Ex Taq (Takara, Japan), and 0.4 µl of 50 × ROX Reference Dye. Primers for the AtUBQ5, AtRAD54, and AtKu70 genes were used according to previous study [27]. The mRNA levels of the AtRAD54 and AtKu70 genes were measured using the Applied Biosystems StepOneTM qRT-PCR device (Life technologies, Singapore) under the following conditions: one cycle of 95°C for 10 s, and then 40 cycles of 95°C for 5 s and 60°C for 31 s. The AtUBQ5 gene was used as an internal control. The final data were the average of 3 independent experiments, with 3 technical replicates for each experiment. 2.5 Treatment of seedlings with exogenous indole-3-acetic acid (IAA) IAA (Sigma Aldrich, St Louis, MO, USA) was dissolved in 100% ethanol, and the stock solution was stored at 4°C. IAA was added into growth medium at a
concentration of 1 nM [28], with less than 0.01% of ethanol (v/v) in the growth medium. Control treatments were applied using equal amounts of ethanol solvent without IAA. After priming irradiation, seedlings were transferred onto the IAA medium for 8 h, and continued to grow for 6 days on IAA medium after challenging irradiation. 2.6 Quantitative analysis and histochemical staining of GUS activity After treatments with gamma-irradiation and/or microgravity, 5-mm long root distal parts were sampled, and quantitative GUS activity was assayed according to protocols described previously [29, 30]. The fluorescence at 455 nm under excitation at 365 nm was measured with a luminescence spectrophotometer equipped with an ELISA plate reader (Spectra Max M2, Molecular Devices, USA). The final data were the average of 15 samples from three independent experiments. For detection of GUS distribution in root tips, the roots of line DR5 were collected after treatments with gamma-irradiation and/or microgravity, and then stained as described previously [31]. 2.7 Statistical Analysis All results are presented as means ± standard deviations. The statistical significance of the experiments was determined by performing Student’s t test. A P value of 0.05 or less was considered significant.
3. Results 3.1 Root growth RAR of A. thaliana
It is well known that one way that plants sense gravity is via physical sedimentation of the statoliths in columella cells in root tips [22]. Thus, in order to investigate the interaction between microgravity and RAR, an experimental model of RAR with plant roots as a checking object was first required. It has been reported that plant root growth is sensitive to ionizing radiation [23, 24]. Therefore, we first tested the relationship between irradiation dose and root growth using 4-day-old seedlings. Low doses of gamma-irradiations (2, 5, 10, and 20 Gy) did not affect the growth of primary roots during various periods after irradiation (in all cases, P > 0.05), as shown in Figure 2A. High gamma-radiation doses (25, 50, 75, 100, 125, and 150 Gy) significantly repressed root growth in a dose-dependent manner (in all cases, P < 0.01), as shown in Figure 2B. It should be noted that root growth within 1 day after irradiation was unchanged (in all cases, P > 0.05), but root growth was suppressed within 2-3 days (in all cases, P < 0.01), and more remarkably within 2-6 days (in all cases, P < 0.01) after irradiation (Figure 2B). Therefore, in the following experiments, root growth within 2-6 days after irradiation was scored, unless otherwise specified. The dose combinations for RAR were further tested. The plants were respectively irradiated with 2, 5, 10, and 20 Gy of gamma-rays (priming doses), and then subjected to 100 Gy of gamma-rays (challenging dose) at an 8 h interval. As shown in Figure 3A, only 10 Gy of priming irradiation significantly alleviated the repressive effect of challenging irradiation on root growth (P < 0.01). In the following experiments, 10 Gy of gamma-irradiation was used as priming dose, and 25, 50, 75, 100, 125, and 150 Gy of gamma-irradiations were used as challenging doses. The RAR was significantly induced by the 10+25 Gy, 10+50 Gy, 10+75 Gy, 10+100 Gy, and 10+125 Gy dose combinations (in all cases, P < 0.05), as shown in Figure 3B. The other time intervals between priming and challenging irradiations (2, 4, 8, 12, 16, and 24 h) were also tested using the 10+100 Gy dose combination. It was shown that the RAR could be induced with time intervals of 4, 8, and 12 h (in all cases, P < 0.01), as shown in Figure 3C. Therefore, in the following experiments, the 10+100 Gy dose combination and 8-h interval were used, unless otherwise specified. 3.2 Effect of modeled microgravity on RAR of root growth
Based on the established experimental model for RAR of root growth, the effect of microgravity on the RAR was further investigated through applying microgravity within the 8 h interval between the priming and challenging irradiations. As shown in Figure 4A, microgravity itself did not interrupt the root growth of naïve plants and plants subjected to 10 and 100 Gy of gamma-irradiations (in all cases, P > 0.05). However, the application of the modeled microgravity significantly prevented the induction of RAR by 10+100 Gy (P > 0.05), as shown in Figure 4B. Moreover, the induction of RAR by the dose combinations of 10+50 Gy and 10+75 Gy was suppressed by the modeled microgravity (in both cases, P > 0.05), as shown in Figure 4C. These results suggest that root growth RAR could be modulated in the modeled microgravity. In order to further strengthen this conclusion, the A. thaliana pgm-1 plants, which are impaired in root gravity sensing due to lack of starch statoliths in the columella cells in the root cap, were also tested. Under normal gravity conditions, the mutant plants also exhibited a suppressed induction of RAR by the10+100 Gy dose combination (P > 0.05), but not by the 10+50 Gy and 10+75 Gy dose combinations (in both cases, P < 0.01), as shown in Figure 4D. 3.3. Time course of interaction between modeled microgravity and RAR In RAR, radio-resistance is developed between priming and challenging irradiations. Here, we investigated the effect of microgravity on the development of RAR. For this purpose, the plants were subjected to microgravity for 0, 2, 4, 6, and 8 h after priming irradiation, respectively, and then exposed to challenging irradiation at 8 h after priming irradiation. The results showed that the induction of RAR was prevented by the application of microgravity within 0-6 and 0-8 h after priming irradiation (in both cases, P > 0.05), but not within 0-0, 0-2, and 0-4 h after priming irradiation (in all cases, P < 0.01), as shown in Figure 5A. Moreover, seedlings were placed in the modeled microgravity at time points of 0, 2, 4, 6, and 8 h after priming irradiation until the application of challenging irradiation, respectively. As shown in Figure 5B, the induction of RAR was significantly suppressed by the application of the modeled microgravity within 0-8 and 2-8 h after priming irradiation (in both cases,
P > 0.05), but not within 4-8, 6-8, and 8-8 h (in all cases, P < 0.01). These results suggest that 6-h duration of microgravity might be required to suppress the development of radio-resistance. The seedlings were also placed in the modeled microgravity for 8 h before priming irradiation. Plant memory of microgravity did not affect the induction of RAR (P < 0.01), as shown in Figure 5C. In another experiment, the plants were subjected to the modeled microgravity for 8 h after treatment with challenging irradiation. Interestingly, the RAR could be suppressed by the later application of microgravity (P > 0.05), as shown in Figure 5D. 3.4 Role of DNA damage repair in RAR of root growth It has been reported that DNA damage repairs play a pivotal role in the induction of RAR [32–34]. In A. thaliana, the non-homologous end joining (NHEJ) and homologous recombination (HR) are two important DNA repair machineries [35], in which the AtRAD54 (HR) and AtKu70 (NHEJ) genes are transcriptionally inducible in response to DNA-damaging agents [36–38]. In order to further know whether microgravity affects the repairing of DNA damage caused by challenging irradiation in root cells, the expressions of the AtKu70 and AtRAD54 genes were detected using qRT-PCR. At 8 h after priming irradiation, just before the application of challenging irradiation, the 10 Gy of gamma-irradiation led to up-regulated expressions of these genes (in both cases, P < 0.01). Interestingly, priming irradiation-induced expressions of AtKu70 and AtRAD54 genes were dramatically down-regulated in the modeled microgravity compared to the 10 Gy irradiation alone (in both cases, P < 0.01) (Figure 6A), suggesting that microgravity might change the priming of DNA damage repairs induced by priming irradiation. The challenging irradiation alone (100 Gy) also led to significantly up-regulated expressions of the AtKu70 and AtRAD54 genes at 2, 12, and 24 h after challenging irradiation (in all cases, P < 0.01), as shown in Figure 6B and C. In normal gravity, their expressions were significantly down-regulated at 12 and 24 h (in all cases, P < 0.01), but not at 2 h (in both cases, P > 0.05) in the irradiation with dose combination of 10+100 Gy compared to the 100 Gy of gamma-irradiation alone, as shown in Figure 6B and C, suggesting that the priming irradiation might promote
the efficiency of DNA damage repair in root cells in response to the challenging irradiation. However, in microgravity, the expression of the AtKu70 gene induced by 10+100 Gy was not down-regulated at 12 h after challenging irradiation compared to the 100 Gy of gamma-irradiation alone (P > 0.05) (Figure 6B), suggesting reduced efficiency of DNA damage repairs in microgravity. 3.5 Role of auxin in modulation of RAR in modeled microgravity It is well accepted that the phytohormone auxin is a key regulator of cell division and elongation [39]. Its polar transport in root tips is regulated by the sedimentation of the statoliths in columella cells [22]. Thus, a question arises as to whether auxin is involved in the interaction between microgravity and RAR of root growth. To address this, seedlings were transferred onto medium containing 1nM of IAA after receiving priming irradiation. The treatment with IAA itself did not affect the root growth of naïve plants or plants irradiated with 10 or 100 Gy of gamma-rays (in all cases, P > 0.05). Interestingly, the induction of RAR of root growth was significantly suppressed in the presence of the IAA (P > 0.05), as shown in Figure 7A. Consistently, the up-regulated expressions of the AtKu70 and AtRAD54 genes by priming irradiation were also significantly suppressed in the presence of IAA (in both cases, P < 0.01), as shown in Figure 7B. The level of auxin in root tips was also measured using DR5 plants, in which GUS activity correlates positively with the auxin level. The auxin level in root tips was not changed by the 10 Gy of priming irradiation (Figure 7C), or 100 Gy of challenging irradiation alone (Figure 7D) (in both cases, P > 0.05). However, irradiation with the 10+100 Gy dose combination led to a decreased level of auxin in the root tips (P < 0.05) (Figure 7D). Interestingly, although the auxin level was not affected in microgravity at 8 h after priming irradiation (P > 0.05) (Figure 7C), the decreased auxin level induced by the 10+100 Gy dose combination was eliminated under the modeled microgravity condition (P > 0.05), as shown in Figure 7D. Similarly, histochemical staining showed an enlarged distribution of auxin in root tips subjected to the 10+100 Gy dose combination in microgravity compared to that in normal gravity, as shown in Figure 7E.
Discussion Space is a complex environment, and many factors, such as radiation particles and microgravity, affect each other at various biological levels [13]. Therefore, their biological effects should be considered in the presence of other factors. Due to the temporal and spatial heterogeneity of space radiation, the RAR is thought to have a potential effect on space crews and the other living organisms carried by spacecraft. Here we present the first evidence that the RAR can be modulated in microgravity. However, the effect of microgravity on RAR was demonstrated only using the root growth of A. thaliana as the biological endpoint; it is unclear whether the RAR of other biological events in different organs or whole organisms, if any, is also modulated by microgravity in the same way. Moreover, in this study the RAR of root growth of A. thaliana was induced by acute exposure to gamma-rays (3.37 Gy/min), and also with higher doses of priming irradiation (10 Gy) and challenging irradiations (25–125 Gy) due to the high radio-resistance of higher plants [40]. Thus, the irradiation pattern and doses used here are not very relevant to a space environment characterized by low-dose and low-fluence radiation. Therefore, in this study, the modulation of RAR by microgravity was demonstrated only in principle, and highly radiosensitive experimental models might be required to demonstrate this phenomenon under more relevant radiation patterns and doses. For higher plants, root growth is related to two zones in the root tip: the meristematic zone (MZ) and the elongation zone (EZ). The root apical meristem (RAM) in the MZ provides new cells for the growing root, and the expansion of root cells in the EZ contributes to primary root growth [39]. In this study, root growth was not affected within 1 day after various doses of gamma-irradiation (Figure 2A), but was significantly suppressed within 2-3 and 2-6 days after irradiation (Figure 2B). It has also been reported that dividing cells are more sensitive to radiation than differentiated cells [41]. Therefore, the repressive effect of gamma-irradiation on root growth might be mainly due to the decreased division of RAM cells. Thus, the
priming irradiation might only up-regulate the radio-resistance of RAM cells, at least in terms of the induction of RAR of root growth. Moreover, it was shown that the radio-resistance induced by priming irradiation persisted only for 12 hours (Figure 3C), whereas the effect of RAR on root growth lasted for 2-6 days after challenging irradiation, during which the RAM has undergone multiple generations of cell divisions. If radio-resistance is induced only in these dividing RAM cells, the effect of RAR on root growth will disappear shortly after these cells enter the EZ. In addition to the dividing cells, the RAM also contains a set of initial cells (stem cells) [39]. The challenging irradiation alone suppressed the root growth within 2–6 days after irradiation (Figure 2B), suggesting that resulting DNA damage in RAM stem cells may persist for a longer time, also possibly transmitted into their progeny cells. Thus, it is likely that the priming irradiation might initiate the radio-resistance in the RAM stem cells that reduces the challenging irradiation-induced persistent DNA damage in these cells. This might be a reasonable explanation for the long-term effect of RAR on root growth. On the other hand, the modeled microgravity inhibited the RAR induced by dose combinations of 10+50 Gy, 10+75 Gy, and 10+100 Gy (see Figure 4B and C), whereas the deficiency of gravity sensation in pgm-1 plants only prevented the induction of RAR by the 10+100 Gy dose combination (see Figure 4D). Like pgm-1 line, the A. thaliana TC7 mutant has been reported to be starch-free and still exhibit root gravitropism [42], indicating that, in addition to the starch-statolith machinery, higher plants also employ other gravity-sensing machinery such as the gravitational pressure model proposed by the Cholodny–Went hypothesis [43]. It is therefore likely that in pgm-1 plants, residual gravity sensing in other cells might attenuate the repressive effect of the absence of starch-statolith machinery on the induction RAR of root growth compared to the whole-body absence of gravity under the modeled microgravity condition. In the present study, treatment with exogenous IAA was shown to inhibit the induction of RAR of root growth (Figure 7A). Moreover, 10 Gy of priming irradiation or 100 Gy of challenging irradiation alone did not affect the level and distribution of auxin in root tips (Figure 7C, D, and E); however, the plants subjected to the 10+100
Gy dose combination exhibited a decreased level of auxin in root tips at 12 h after challenging irradiation (Figure 7D and E). These results indicate that auxin might play a regulatory role in the induction of RAR of root growth. Auxin is one of key regulators of cell division in the MZ of roots, and the genes in auxin biosynthesis are also expressed specifically in the root stem cell niche [44–46]. Disturbances in auxin level and distribution can change the cycle phases of RAM cells [39], to which the radio-sensitivity of cells is closely related. It has been reported that the induction of RAR is largely dependent on radio-sensitivity [32]. Moreover, DNA damage repairs also play a critical role in the induction of RAR [32–34]. It has been reported that the expression of the Ku gene in hypocotyls of Vigna radiate is regulated by auxin [47]. In this study, the priming irradiation-induced expressions of the AtKu70 and AtRAD54 genes were suppressed in the presence of IAA (Figure 7B). Therefore, it is likely that auxin might affect the induction of RAR of root growth through changing the radio-sensitivity and/or DNA repair of RAM cells. In plant root tips, polar transportation of auxin is regulated by gravity via the starch-statolith machinery [22]. For the 10+100 Gy dose combination, a different level and distribution of auxin in the root tips was also observed at 12 h after challenging irradiation in microgravity compared to that in normal gravity (Figure 7D and E). Therefore we reason that microgravity suppresses the induction of RAR of root growth through interfering with the level and distribution of auxin in root tips. In addition to auxin, other plant hormones such as abscisic acid (ABA) and gibberellic acid (GA) also take part in the regulation of cell division in the MZ [39] and in DNA damage repairs [48, 27]. However, due to the absence of evidence about the regulations of microgravity on their transportation and signaling transduction, it does not appear that they play regulatory roles in interaction between microgravity and RAR of root growth. However, it is likely that microgravity could regulate them through cross-talk of signaling between auxin and these plant hormones [49, 50].
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments We thank Prof. Guifoyle and NASC for their generous provision of the various A. thaliana seeds. This work was supported by the National Science Fund of China (11575233, 11275230), Natural Science Fund of Anhui Province (1408085MKL51) and International S&T Cooperation Program of China (2015DFR30920).
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Legends Fig.1 Schematic representation of gamma-irradiation and microgravity simulation of Arabidopsis thaliana.
Fig.2 The dose relationship of root growth of A. thaliana seedlings. A) Root growth at indicated periods after 2–20 Gy of gamma-irradiation; B) Root growth at indicated periods after 25–150 Gy of gamma-irradiation. Results are means ± SD (n = 3, t test, ** P < 0.01).
Fig.3 Induction of RAR of root growth by combined gamma-irradiations. A) Root growth of seedlings subjected to 2, 5, 10, and 20 Gy of priming gamma-irradiations, and 100 Gy of challenging gamma-irradiation; B) Root growth of seedlings subjected to 10 Gy of priming gamma-irradiation and 25, 50, 75, 100, 125, and 150 Gy of challenging gamma-irradiations; C) Effect of time intervals between priming and challenging irradiations on the induction of RAR. Results are means ± SD (n = 3, t test, * P < 0.05, ** P < 0.01).
Fig.4 Modulation of microgravity on the RAR of root growth. A) Effect of microgravity on the root growth of naïve or irradiated seedlings; B) Effect of modeled microgravity on the RAR induced by 10+100 Gy dose combination; C) Effect of modeled microgravity on the RAR induced by 10+50 Gy and 10+75 Gy dose combinations; D) The induction of RAR in pgm-1 plants deficient in root gravity sensing. Results are means ± SD (n = 3, t test, ** P < 0.01).
Fig.5 Time course of interaction between RAR and microgravity. A) The induction of RAR in seedlings placed in modeled microgravity for the indicated durations after priming irradiation; B) The induction of RAR in seedlings placed in modeled microgravity at time points of 0, 2, 4, 6, and 8 h after priming irradiation until 8 h after priming irradiation; C) The induction of RAR in seedlings placed in modeled
microgravity for 8 h before priming irradiation; D) The induction of RAR in seedlings placed in modeled microgravity for 8 h after challenging irradiation. Results are means ± SD (n = 3, t test, ** P < 0.01).
Fig.6 Expressions of DNA repair genes in roots after treatments with gamma-irradiation and/or modeled microgravity. A) Effect of microgravity on mRNA levels of the AtKu70 and AtRAD54 genes at 8 h after priming irradiation; B) mRNA level of the AtKu70 gene at 2, 12, and 24 h after challenging irradiation under the normal gravity (NG) and modeled microgravity (MG); C) mRNA level of the AtRAD54 gene at 2, 12, and 24 h after challenging irradiation in the normal gravity (NG) and modeled microgravity (MG). Results are means ± SD (n = 3, t test, ** P < 0.01).
Fig.7 Effect of the phytohormone auxin on the induction of RAR. A) Effect of exogenous IAA on the induction of RAR of root growth; B) mRNA levels of the AtKu70 and AtRAD54 genes at 8 h after priming irradiation in the presence of IAA; C) Level of auxin in root tips at 8 h after priming irradiation; D) Level of auxin in root tips at 12 h after challenging irradiation; E) Histochemical staining of GUS activity in DR5 seedlings at 12 h after challenging irradiation. Results are means ± SD (n = 3 for analysis of root growth, n = 15 for analysis of GUS activity, t test, * P < 0.05, ** P < 0.01).