Factors Affecting the Radiosensitivity of Hexaploid Wheat to -Irradiation: Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)

Abstract

Understanding the radiosensitivity of plants, an important factor in crop mutation breeding programs, requires a thorough investigation of the factors that contribute to this trait. In this study, we used the highly radiosensitive wheat (Triticum aestivum L.) variety HY1 and J411, a γ-irradiation-insensitive control, which were screened from a natural population, to examine the factors affecting radiosensitivity, including free radical content and total antioxidant capacity, as well as the expression of TaKu70 and TaKu80 (DNA repair-related genes) as measured by real-time PCR. We also investigated the alternative splicing of this gene in the wild-type wheat ecotype by sequence analysis. Free radical contents and total antioxidant capacity significantly increased upon exposure of HY1 wheat to γ-irradiation in a dose-dependent manner. By contrast, in J411, the free radical contents exhibited a similar trend, but the total antioxidant capacity exhibited a downward trend upon increasing γ-irradiation. Additionally, we detected dose-dependent increases in TaKu70 and TaKu80 expression levels in γ-irradiated HY1, while in J411, TaKu70 expression levels increased, followed by a decline. We also detected alternative splicing of TaKu70 mRNA, namely, intron retention, in HY1 but not in J411. Our findings indicate that γ-irradiation induces oxidative stress and DNA damage in hexaploid wheat, resulting in growth retardation of seedlings, and they suggest that TaKu70 may play a causal role in radiosensitivity in HY1. Further studies are required to exploit these factors to improve radiosensitivity in other wheat varieties.

Full text

RESEARCH ARTICLE
Factors Affecting the Radiosensitivity of
Hexaploid Wheat to γ-Irradiation:
Radiosensitivity of Hexaploid Wheat
(Triticum aestivum L.)
Bing Han
1,2☯
, Jiayu Gu
2☯
, Linshu Zhao
2
, Huijun Guo
2
, Yongdun Xie
2
, Shirong Zhao
2
,
Xiyun Song
1
, Longzhi Han
2
, Luxiang Liu
2
*
1Academy of Life Science, Qingdao Agricultural University, Qingdao, China, 2Institute of Crop Sciences,
Chinese Academy of Agricultural Sciences /National Key Facility for Crop Gene Resources and Genetic
Improvement /National Center of Space Mutagenesis for Crop Improvement, Beijing, China
☯These authors contributed equally to this work.
*liuluxiang@caas.cn
Abstract
Understanding the radiosensitivity of plants, an important factor in crop mutation breeding
programs, requires a thorough investigation of the factors that contribute to this trait. In this
study, we used the highly radiosensitive wheat (Triticum aestivum L.) variety HY1 and J411,
aγ-irradiation-insensitive control, which were screened from a natural population, to exam-
ine the factors affecting radiosensitivity, including free radical content and total antioxidant
capacity, as well as the expression of TaKu70 and TaKu80 (DNA repair-related genes) as
measured by real-time PCR. We also investigated the alternative splicing of this gene in the
wild-type wheat ecotype by sequence analysis. Free radical contents and total antioxidant
capacity significantly increased upon exposure of HY1 wheat to γ-irradiation in a dose-
dependent manner. By contrast, in J411, the free radical contents exhibited a similar trend,
but the total antioxidant capacity exhibited a downward trend upon increasing γ-irradiation.
Additionally, we detected dose-dependent increases in TaKu70 and TaKu80 expression
levels in γ-irradiated HY1, while in J411, TaKu70 expression levels increased, followed by a
decline. We also detected alternative splicing of TaKu70 mRNA, namely, intron retention, in
HY1 but not in J411. Our findings indicate that γ-irradiation induces oxidative stress and
DNA damage in hexaploid wheat, resulting in growth retardation of seedlings, and they
suggest that TaKu70 may play a causal role in radiosensitivity in HY1. Further studies are
required to exploit these factors to improve radiosensitivity in other wheat varieties.
Introduction
The induced mutation technique, an important application of nuclear technology in agricul-
ture, has significantly contributed to crop germplasm enhancement and new mutant variety
PLOS ONE | DOI:10.1371/journal.pone.0161700 August 23, 2016 1/15
a11111
OPEN ACCESS
Citation: Han B, Gu J, Zhao L, Guo H, Xie Y, Zhao S,
et al. (2016) Factors Affecting the Radiosensitivity of
Hexaploid Wheat to γ-Irradiation: Radiosensitivity of
Hexaploid Wheat (Triticum aestivum L.). PLoS ONE
11(8): e0161700. doi:10.1371/journal.pone.0161700
Editor: Arthur J. Lustig, Tulane University Health
Sciences Center, UNITED STATES
Received: April 3, 2016
Accepted: August 10, 2016
Published: August 23, 2016
Copyright: © 2016 Han et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by the National
973 Program: grant no. 2014CB138101, the National
Key Technology R&D Program of China: grant
no.2014BAA03B04, Core Research Budget of the
Non-profit Governmental Research Institutions (ICS,
CAAS), and the National Natural Science Foundation
Research Program: grant no. 11305261.
Competing Interests: The authors have declared
that no competing interests exist.

development. Studies investigating radiation sensitivity have played prominent roles in reveal-
ing the mechanism of induced mutations. Differences in the radiosensitivity of various crops
have been investigated [1–3]. However, the molecular mechanisms responsible for radiosensi-
tivity are currently unclear, making research in this area vital. Investigations in animals and
human have revealed a link between cell radiosensitivity and variations in free radicals, oxida-
tive stress, and DNA repair mechanisms [4–10]. Therefore, it is important to further explore
radiosensitivity and its molecular determinants. Such information would be useful for predict-
ing and modulating radiosensitivity.
Organisms exposed to irradiation are induced to produce reactive oxygen species (ROS),
which can give rise to DNA double-strand breaks (DSBs), which in turn can affect proteins.
However, some plants, during the long process of evolution, have developed the ability to with-
stand ionizing radiation (IR) stress. Under IR stress, such plants can activate ROS removal sys-
tems and DNA repair systems. The former includes enzyme and non-enzyme based systems.
The enzyme systems utilize amongst others, SOD, GPX and catalase to quickly remove ROS.
Non-enzymatic systems utilize, amongst others, the surfhydryl of glutathione (GSH), ascorbic
acid and the carotenoqesls to quench oxyradicals [10–16]. Currently, it is widely accepted that
oxidative stress is involved in the pathogenesis of many diseases, including various cancers and
degenerative disorders in animals [17]. In our study, we assess the effects of ROS on hexaploid
wheat, as well as its total antioxidative capacity (T-AOC). DSBs caused by IR is disastrous to
both cells and organisms. It can trigger chromosomal rearrangements, aneuploidy and the loss
of segments of chromosomes. If not followed by rapid and successful repair, cell and whole
organism death can be infamous result [18–21]. In eukaryotes, Non-homologous end-joining
(NHEJ) is the most important DSB repair pathway [22–25]. ku70/ku80 heterodimers recognize
DSBs and bind to them, forming complexes. These recruit DNA-dependent protein kinase cat-
alytic subunits which initiate the NHEJ repair process [26–29], Errors in or DNA mutations
involving this process can lead to failure of repair and so increase the sensitivity of the organ-
ism to ionizing radiation [30]. In our study, defects in the TaKu70 gene may be one reason for
the high sensitivity of our HY1 strain. Under normal circumstances, alternative splicing of pre-
mature RNA is an important process utilized by eukaryotes to produce all kinds of protein
forms from a single gene [31–34]. This can enhance protein diversity and regulates some pro-
cess in plants [35–37]. Some studies have suggested that the alternative splicing of single genes
can create small amounts of protein isoforms in plants. In rice, approximately 68.3% of genes
create only one isoform. However, among all splicing types, intron retention is common [38–
40]. In wheat, investigations are ongoing. However, with regards mechanisms and function,
there seem to be few surprises [41]. Evidence suggests that weak splice sites, shorter introns
and lower density splicing enhancers intron retention [42–43]. Therefore, their retention sug-
gests the occurrence of missplicing, caused by problems with the splicing machinery [44]. In
addition, alternative splicing regulates the expression of certain critical genes [45–48].
Any defect of alternative splicing can cause severe problems to the organism as a result of
changes in their protein composition. Frame shifts are one consequence of miss-splicing [36].
In Arabidopsis 42% of fame shift events create a premature stop codon; in rice, the frequency is
36% [32]. In our study, intron retention was found in the TaKu70 gene. This may reflect splic-
ing errors in the pre-mRNA splicing process, prematurely stopping the expression of TaKu70.
Ultimately, defective TaKu70 protein might be produced.
Phenotype studies of ku70-defects in DNA repair mechanisms have been carried out in
fungi, yeasts and animals [49]. Disruption of Ku70 in mouse embryonic stem cells results in
markedly increased sensitivity to ionizing radiation [50]. Ku70-deficient mice are approxi-
mately half the size of control mice, and their fibroblasts are sensitive to ionizing radiation and
display premature senescence associated with the accumulation of nondividing cells [51]. In
Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)
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Arabidopsis, ku mutants can be associated with telomere elongation [52]. In our study,
TaKu70 defective wheat was highly sensitivity to γradiation. This produced severe phenotypic
changes compared to the controls.
We previously cloned Ku70 and Ku80 in wheat, which were designated TaKu70 and
TaKu80, respectively [53–55]. The functions of the two genes, as well as the encoded protein,
have been investigated [55]. We previously selected the hexaploid wheat variety HY1, which
exhibited the highest sensitivity to γ-irradiation among the 63 wheat genotypes examined,
whereas wheat variety J411 exhibited insensitivity to γ-irradiation[56]. In this study, the
increased radiosensitivity of this variety allowed us to analyze the combined effects of an exoge-
nous agent and IR on plants[14]. The results of this study may help shed light on the mecha-
nism underlying radiosensitivity in wheat.
Materials and Methods
γ-irradiation and free radical contents assay
The moisture content of the dry seeds of HY1 and J411 was balanced with glycerin and water
ratio of 1:1 to up to 13% and they were then irradiated by gamma rays at dosages of 100, 150
and 250 Gy (7 Gy/min; The Department of Radiation at Peking University, Beijing, China).
The free radical contents under each dosage were immediately examined using electron spin
resonance apparatus (ESR, E-Scan, BRUKER, SC0340, Germany), and various parameters
were obtained, namely Food, Marker, g1-value, g2-value, Frequency. The seeds under each
dosage were randomly examined five times. The Food parameters values, which represent vari-
ations in free radicals, were normalized using the formula: Foodn = Food × 400000 / Marker.
Foodn values can take the place of relative free radical contents values.
Plant materials and cultivation
All wheat seeds were kindly provided by the Chinese Academy of Agricultural Sciences. Prior
to placing them on a hydroponics shelf for seedling growth, the dried seeds were immersed in
distilled water for germination for 16 hours with a rate of 50 seeds/15 mL water (using three
replicates per dosage). Germinated seeds were placed on a hydroponics shelf under controlled
conditions (16 h light/12 h dark, temperature: 25°C, relative humidity: 78%). Leaves were har-
vested on the day 5 for DNA and RNA extraction.
Total antioxidative capacity (T-AOC) assay
The irradiated seeds were soaked in distilled water for 16 h (using three replicates per dosage)
and transferred to the germination apparatus under constant conditions. Leaf samples (0.05 g)
were collected on day 5, instantly placed into 2 mL EP tubes containing 500 μL 0.9% normal
saline and ground into a powder using a tissue grinder apparatus (30 Hz/s, 60 s). The samples
were centrifuged for 300 s at 5,000 rpm, and 300 μL of supernatant was transferred to a fresh
1.5 mL centrifuge tube (on ice). The T-AOC assay was carried out using a T-AOC reagent kit,
and the OD value at 520 nm was measured following the manufacturer’s instructions using the
formulaT −AOC = (ODu −ODc) / 0.01 / 30 N / CProt.
DNA isolation, PCR amplification and sequencing
Genomic DNA was obtained from a pool of DNA extracted from ten HY1 and J411 leaves
using a Caliper workstation and a DNA Secure Plant Kit. To determine the source of the
retained fragment in the cDNA, specific primers were designed (25AER: 5’GGCACTGCTG
CGTAAAGG 3’, 25AEF: 5’TCACCAGCAGATGGCACG 3’) based on the A, B and D genome
Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)
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sequences [55]. For amplification and sequencing of the 879 bp gene containing the 133 bp
retention fragment, primers were designed approximately 20 to 50 bp upstream and down-
stream of the coding region. For analysis of the region, the fragments of accessions were ampli-
fied using the proofreading polymerase Phusion (Finnzymes) and subjected to TA clone prior
to sequencing. The positive clones were directly used for sequencing.
RNA isolation, cDNA synthesis and real-time PCR
RNA was isolated with TRNzol
A+
solution. For each accession, three biological replicates were
performed, and 1 μg RNA was reversely transcribed using a Transcriptor High Fidelity First-
strand cDNA Synthesis Kit (Roche, Version 6, Germany). For the TaKu70 gene, 10 accessions
were selected for RT-PCR and TA cloning. For each accession, 72 positive clones were selected
for sequencing and analyses. Primary analyses revealed alternative splicing in the cDNA frag-
ment from the HY1 accessions, which was confirmed using a validation protocol. Data analy-
sis, sequence alignment and were performed with BioEdit version 7.05 for each accession, and
all variable sites were checked manually during the construction of a sequence contig. All
sequences were manually aligned to the reference sequence. RNA was reversely transcribed
and used at 1 μg per real-time-PCR run in a 10 μL reaction volume using SsoFast™EvaGreen
Supermix and a C1000™thermal cycler; each biological replicate included three technical repli-
cates. Expression was normalized to the Actin and 18s genes. The primer sequences are shown
in S1 Table. A CFx96™Real-time System was used for analysis.
Data analysis
Statistical analysis was performed using SPSS 16.0, with one-way ANOVA performed to test
the significance of differences when more than two groups were involved. Values were consid-
ered significantly different if (P <0.05). Comprehensive data analysis was performed using
Heml1.0 software.
Results
Variation in free radical contents
To investigate the oxidative stress caused by gamma irradiation, we measured the free radical
contents in HY1 wheat and the control variety J411 in response to various dosages of IR (Fig
1). The relative free radical content increased significantly in a dose-dependent manner. At 250
Gy, the relative free radical levels were significant increased in these plants, reaching more than
3.0-fold control dosage levels (under 0 Gy treatment). The free radical contents were more
than 2.0-fold control levels under 100 Gy treatment and 2.5-fold control levels under 150 Gy
treatment. These levels were significantly higher in the treatment groups than in the control
dosage group (p <0.05). In the control variety J411, the relative free radical contents increased
in a dose-dependent manner, but the upward trend was slower. The basal levels of free radicals
in HY1 and J411 were similar.
Total antioxidant capacity (T-AOC)
To analyze the roles of enzymatic and non-enzymatic components in HY1 and the control
variety J411, namely, the T-AOC of these plants, we used a total antioxidant capacity kit to
measure OD values at 520 nm, which revealed T-AOC values under each dosage of IR (Fig 2).
In HY1, the T-AOC values increased significantly with increasing gamma irradiation dosage.
Under 250 Gy treatment, T-AOC reached more than two-times the levels measured under the
control dosage levels. At lower doses, however, increases in T-AOC values were drastically
Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)
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lower, approaching the values of the control dosage group, The basal T-AOC was higher in the
control variety J411 than in HY1, whereas at 100 Gy and 150 Gy, these values were consistent
in the two varieties, and at 250 Gy, this value was lower in J411 than the HY1.
DNA repair-related gene expression
To investigate changes in the expression of the DNA repair-related genes TaKu70 and TaKu80
in response to gamma irradiation, we monitored the expression of these genes in IR-treated
Fig 1. Effect of gamma irradiation on free radical levels. The X-axis represents the treatment dosage, including 0 Gy, 100 Gy, 150 Gy and 250 Gy.
The Y-axis represents the free radical levels. Dark gray bars indicate free radical contents in HY1, light gray bars represent free radical contents in the
J411 variety. Significant differences between treatment groups and the control groups in the HY1and J411 variety were analyzed by spass 16.0
(P <0.05).
doi:10.1371/journal.pone.0161700.g001
Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)
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HY1 and the control variety J411 (Fig 3E and 3F). In HY1, under high doses of gamma irradia-
tion, both genes were significantly induced, especially TaKu70; at 250 Gy, the expression level
of this gene was more than three-times that of the control(0Gy). However, under 100 Gy treat-
ment, both genes were only slightly induced. The phenotypes of HY-1 in response to 0, 100,
150, and 250 Gy gamma irradiation are shown in Fig 3B, Seedling height and root length signif-
icantly decreased with increasing gamma irradiation dosage. while in the control variety J411,
TaKu70 was significantly induced at dosages of 100 Gy and 150 Gy. At 100 Gy, the expression
level of this gene was more than two-times that of the control dose (0 Gy). Taku80 expression
Fig 2. Effect of gamma irradiation on T-AOC in HY1 and J411 wheat. The X-axis represents the treatment dosage, including 0 Gy, 100 Gy,
150 Gy and 250 Gy. The Y-axis represents T-AOC levels. Dark gray bars represent T-AOC values in HY1, and light gray bars represent those in
J411. Significant differences between treatment groups and the control groups in the HY1and J411 variety were analyzed by spass 16.0
(P <0.05).
doi:10.1371/journal.pone.0161700.g002
Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)
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levels were consistent with the levels detected in HY1. The phenotypes of J411 in response to
IR are shown in Fig 3A. Seedling height and root length decreased slightly with increasing
gamma irradiation dosage. Histogram analysis of seedling height and root length in HY1 and
the control variety J411 is shown in Fig 3C and 3D. Seedling height decreased significantly with
Fig 3. Effect of gamma irradiation on DNA repair-related genes TaKu70 and TaKu80 and plant
phenotypes.(A, B) Photographs of HY1 and J411 plants under different dosages of γ-irradiation. Seedling
height and root length decreased significantly with increasing gamma irradiation dosage more quickly in HY1
than in J411. (C, D) Histogram analysis of variation rate of root length and seedling height in HY1 and J411.
The X-axis represents the treatment dosage, including 0 Gy, 100 Gy, 150 Gy and 250 Gy. The Y-axis
represents the variation rate of root length and seedling in HY1 and J411. Significant differences were
analyzed by spass 16.0 (P <0.05) (E, F)The X-axis represents the treatment dosage, including 0 Gy, 100 Gy,
150 Gy and 250 Gy. The Y-axis represents Taku70 gene expression level. Dark gray bars indicate Taku70
and Taku80 gene expression values in HY1, and light gray bars indicate those in J411. Significant differences
between treatment groups and the control groups in the HY1and J411 variety were analyzed by spass 16.0
(P <0.05).
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increasing gamma irradiation dosage in both HY1 and J411, but the reduction was slower in
J411 than in HY1 (Fig 3C and 3D)
Intron retention in TaKu70
To explore the transcriptional regulation of TaKu70, which affects the radiosensitivity of HY1
and the control variety J411, we cloned TaKu70 cDNA sequences (S2 Fig) from both varieties
and subjected the mRNA sequences to alignment (Fig 4A). In HY1, cDNA sequence alignment
(S2 Fig) showed that a 133 bp fragment located between 601 bp and 733 bp was retained in
its mRNA sequence. A comparison between HY1 TaKu70 cDNA and the wild-type TaKu70
sequence (S1 Fig) showed that the retained 133 bp fragment was derived from the sixth intron
(67 bp) located between 2,544 bp and 2,610 bp and the seventh intron (66 bp) located between
Fig 4. Analysis of the alternative splicing, namely, intron retention. (A) The first region highlighted in gray represents the sixth exon., and the second
represents the eighth exon. Ku70-cDNA and the ku70 –A sequence were obtained from the Chinese spring variety cloned by our team as the standard
comparison sequence. (B) J411-1, J411-14, J411-24 s equence, and so on represent 19 clone replicates from J411.
doi:10.1371/journal.pone.0161700.g004
Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)
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2,750 bp and 2,815 bp in the A genome (Fig 4A). In J411, 19 clone replicates were carried out,
and is no retention was detected in its mRNA (Fig 4B,S4 Fig).
Amino acid analysis of encoded TaKu70 protein
As shown in Fig 5, normal TaKu70 mRNA encodes a functional protein, TaKu70, containing
626 amino acid residues. However, TaKu70 exhibiting intron retention encodes a non-func-
tional TaKu70 protein of only 200 amino acid residues (S3 Fig). Whether this protein is actu-
ally produced and functional remains unclear.
Integrated analysis of all data
Heml1.0 software was used to construct a heat map of all of the data. As shown in Fig 6, the
clustering results suggest that the phenotypic variation is related to TaKu70 expression and
T-AOC levels. Phenotypic variation is a standard measure of radiosensitivity. In the J411 vari-
ety, the A-TOC levels, TaKu70 expression levels, phenotypes and radicals levels were clustered
together then TaKu80 expression levels. On the Y-axis, 100 Gy and 150 Gy were clustered
together (Fig 6A). In the HY1 variety, TaKu70 and TaKu80 expression levels, T-AOC and radi-
cals levels were clustered together, which ultimately influence the phenotype. On the Y-axis, 0
Gy and 100 Gy were clustered then150 Gy (Fig 6B).
Discussion
The molecular mechanisms responsible for radiosensitivity are currently unclear, making this
topic a vital area of research. In the current study, we explored the molecular determinants of
radiosensitivity. We chose the hexaploid wheat variety HY1 and the variety J411 as a control:
the former was previously found to be the most susceptible variety to gamma irradiation and
the latter is not radiosensitive to gamma irradiation among the 63 wheat genotypes examined
[55–56]. To date, several studies examining the effects of gamma irradiation have been carried
out in terrestrial organisms and some aquatic vertebrates [57–59]. Some studies suggest that
Fig 5. The effect of intron retention on the encoded protein. TaKu70 encodes a 626 amino acid protein. Gray
highlighting represents the protein encoded by the mRNA harboring intron retention in HY1.
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different levels of susceptibility to radiation between organisms are likely due to differences in
DNA content, repair processes, and cell cycle kinetics [60]. In the current study, we found that
the high radiosensitivity of HY1 may be linked to the DNA repair process and A-TOC levels
(Fig 6). However, the underlying mechanisms that function in organisms upon gamma irradia-
tion have not been investigated in detail.
Free radicals play an important role in radiosensitivity. We therefore assessed the free radi-
cals levels of the control variety J411 and HY1, finding that both of these levels increased upon
exposure to radiation, but the increase was more rapid in HY1 than in J411 (Fig 1). This result
suggests that under the same γ-irradiation conditions, J411 may produce fewer free radicals
than HY1. The accumulation of free radicals can lead to DSBs and a variety of molecular effects,
including preventing cell division, aging and apoptosis [61]. Organisms utilize enzymatic and
non-enzymatic systems to counteract the effects of free radicals in an attempt to maintain cellu-
lar homeostasis [62]. We therefore examined the T-AOC of enzymatic and non-enzymatic anti-
oxidants using a total antioxidant capacity kit. HY1 had lower basal A-TOC levels than J411.
The HY1 cells exhibited lower T-AOC under lower doses of gamma irradiation (100 Gy and
150 Gy), but quickly increased at high doses (250 Gy). While in J411, the basal T-AOC level was
Fig 6. Heat map analysis of all data. The abscissa represents free radical levels, A-TOC levels, seedling
height, root length, TaKu70 and TaKu80 expression levels. The ordinate represents different dosages of γ-
irradiation. The primary data were LOG
2
transformed using Heml1.0 software. The color variation represents
different values.
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higher than that of HY1, and the T-AOC levels decreased with increasing irradiation (Fig 2).
The cause of this difference is currently unclear. Perhaps the high dose of γ-irradiation induced
some enzymatic activities, and the primordial T-AOC substance can remove the induced
T-AOC substance in the J411 variety. These results all suggest that enzymatic and non-enzy-
matic antioxidants play main role in resisting irradiation stress in the two varieties. Additionally,
the enzymatic and non-enzymatic system comprises several components, such as SOD, GSH,
CAT, GR, GST, GPX, and so on [63]. The total antioxidant enzyme activities displayed in Fig 2
may not represent the effect of each individual enzyme. However, interestingly, the expression
of the DNA repair-related gene TaKu70 (Fig 3E) dramatically increased under 100 Gy gamma
irradiation and then began to decline in J411. In addition, the TaKu70 mRNA sequence lacked a
retention segment (Fig 4B), suggesting that J411 TaKu70 mRNA encodes the normal protein
and has completed the DNA repair progress, which may be an important cause of the low radio-
sensitivity of this variety. In HY1, TaKu70 was significantly upregulated at 150 Gy and 250 Gy.
However, segment retention occurred in the mRNA of HY1, which may encode a nonfunctional
protein (Fig 4A). This segment retentionb may be an important cause of the radiosensitivity of
HY1. These results suggest that the DNA repair system may greatly contribute to the varied
radiosensitivity of J411 and HY1. The different A-TOC levels in the two varieties likely contrib-
utes to this difference as well. Comprehensive heat map analysis of all of the data (Fig 6A and
6B) helped confirm the above-mentioned results. Some researchers have proposed that gene
transcription levels are a reliable early signal for detecting physiological changes under environ-
mental stress [64–65]. Elucidating the expression patterns of specific genes would be helpful for
better understanding the underlying molecular mechanisms of radiosensitivity upon γ-irradia-
tion. Ku70 and Ku80 are induced by γ-irradiation in a dose-dependent manner in the marine
copepod Paracyclopina nana [63]. In human, Ku70, and Ku80 are key components of the DSBs
repair process, as they ligate the broken ends of DNA in the absence of homologous templates
[66]. Thus, the increased expression of TaKu70 and TaKu80 in gamma-irradiated HY1 and
J411 implies that these genes are closely related to the enhanced DNA repair process that func-
tions to recover oxidative stress-induced cellular damage in these plants.
RNA sequencing confirmed that intron retention occurred in TaKu70 mRNA (Fig 4A)in
HY1, which may also help explain the increased radiosensitivity of this genotype. The 133 bp
fragment retained in the mRNA would lead to the production of a 200 amino acid, non-func-
tional TaKu70 protein (Fig 5). Thus, DNA repair of DSBs would be weakened or inhibited,
which may contribute to the high radiosensitivity of the HY1 variety. Disruption of Ku70 in
mouse embryonic stem cells results in severely increased sensitivity to ionizing radiation [50].
The aborted DNA repair process might cause the phenotypic variation observed in HY1 (Fig
3A), Ku70-deficient mice are approximately 50% the size of the control [51], the phenotypic
effects of different doses of radiation were reconfirm in our experiments (Fig 3A and 3B). How-
ever, our knowledge of the physiological relevance of this important post-transcriptional regu-
latory mechanism in plants is quite limited. The current study provides functional evidence
that alternative splicing plays a important role in plant responses to environmental stress [41].
Our data open up the possibility for further study of a probable link between alternative splic-
ing and hypersensitivity to γ-ionizing radiation in plants,
In summary, our results suggest that there is a correlation between radiosensitivity and
intron retention, as well as activation of the antioxidant and DNA repair systems, in HY1. No
previous studies have investigated the radiosensitivity mechanism in other plants. In this
study, we found that the antioxidant and DNA repair systems were induced by gamma irradia-
tion to mitigate damage from free radicals. Additionally, alternative splicing, namely intron
retention, might contribute to the radiosensitivity of HY1. Further evidence is needed to con-
firm the correlation between radiosensitivity and intron retention in hexaploid wheat.
Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)
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Supporting Information
S1 Fig. The comparison of HY1 TaKu70 cDNA, Ku70-A and TaKu70 cDNA. Ku70-A
genome and Taku70cDNA was cloned from the Chinese spring. HY1Taku70 cDNA was
cloned from HY1.
(PDF)
S2 Fig. The comparsion of HY1 TaKu70 cDNA and TaKu70 cDNA. 133 bp fragment located
between 601 bp and 733 bp was retained in the mRNA sequence in HY1.
(PDF)
S3 Fig. Amino acid analysis of encoded TaKu70 protein. Taku70 encodes a 626 amino acid
residues protein. The detained mRNA encoded 200 amino acid residues protein.
(PDF)
S4 Fig. The original sequences of the 19 clone repeats of Taku70 gene mRNA from J411
variety.
(PDF)
S1 Table. primer Squence used in the Quantitative Real-time PCR.
(PDF)
Acknowledgments
We would like to thank all the members who involved in the experiment in this study, The
authors deeply appreciate all the technicians for their assitance in experiments. We particularly
thank the reviewers to give us the constructive suggestions about the manuscript.
Author Contributions
Conceptualization: LL XS BH JG LH.
Data curation: LL BH.
Formal analysis: BH.
Funding acquisition: LL JG.
Investigation: BH JG.
Methodology: BH JG HG.
Project administration: BH LZ.
Resources: LL LZ HG YX SZ.
Software: JG.
Supervision: LL XS YX SZ LH.
Validation: LL XS.
Visualization: BH JG.
Writing –original draft: BH.
Writing –review & editing: YS BH.
Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)
PLOS ONE | DOI:10.1371/journal.pone.0161700 August 23, 2016 12 / 15

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