Photodamage repair pathways contribute to the accurate maintenance of the DNA methylome landscape upon UV exposure.

Plants are exposed to the damaging effect of sunlight that induces DNA photolesions. In order to maintain genome integrity, specific DNA repair pathways are mobilized. Upon removal of UV-induced DNA lesions, the accurate re-establishment of epigenome landscape is expected to be a prominent step of these DNA repair pathways. However, it remains poorly documented whether DNA methylation is accurately maintained at photodamaged sites and how photodamage repair pathways contribute to the maintenance of genome/methylome integrities. Using genome wide approaches, we report that UV-C irradiation leads to CHH DNA methylation changes. We identified that the specific DNA repair pathways involved in the repair of UV-induced DNA lesions, Direct Repair (DR), Global Genome Repair (GGR) and small RNA-mediated GGR prevent the excessive alterations of DNA methylation landscape. Moreover, we identified that UV-C irradiation induced chromocenter reorganization and that photodamage repair factors control this dynamics. The methylome changes rely on misregulation of maintenance, de novo and active DNA demethylation pathways highlighting that molecular processes related to genome and methylome integrities are closely interconnected. Importantly, we identified that photolesions are sources of DNA methylation changes in repressive chromatin. This study unveils that DNA repair factors, together with small RNA, act to accurately maintain both genome and methylome integrities at photodamaged silent genomic regions, strengthening the idea that plants have evolved sophisticated interplays between DNA methylation dynamics and DNA repair.

Identification and Characterization of γ-Ray-Induced Mutations in Rice Cytoplasmic Genomes by Whole-Genome Sequencing.

Chloroplasts and mitochondria are semi-autonomous organelles and have their own genomes (cytoplasmic genomes). Physical radiations (e.g., γ-rays) have been widely used in artificial mutation induction for plant germplasm enhancement and for breeding new cultivars. However, little is known at the genomic level about which kind of cytoplasmic mutations and/or characteristics could be induced in plants. The present study aimed to investigate the type, number, and distribution of inheritable cytoplasmic mutations induced by γ-rays in rice (Oryza sativa L.). Six plants were selected from the 2nd generation (M2) populations after γ-ray (137Cs) irradiation of the rice cultivar Nipponbare, 2 each for the 3 irradiation doses (150, 250, and 350 Gy), and their genomes were sequenced on an Illumina platform. Together with the whole-genome sequencing data of 3 external Nipponbare control plants, single-base substitutions (SBSs) and insertions/deletions (InDels) in chloroplast (cp) and mitochondrial (mt) genomes were identified and analyzed in-depth using bioinformatic tools. The majority of SBSs and InDels identified were background mutations in the 6 M2 plants, and the number of induced mutations varied greatly among the plants. Most induced mutations were present in a heterogeneous state, reflecting the fact that multiple cp and mt copies existed in the progenitor cells. The induced mutations were distributed in different genomic regions in the 6 M2 plants, including exonic regions, but none of them was predicted to cause nonsynonymous mutations or frameshifts. Our study thus revealed, at the genomic level, characteristics of cytoplasmic mutations induced by γ-rays in rice.

RNA-seq analysis of the transcriptional response to blue and red light in the extremophilic red alga, Cyanidioschyzon merolae.

Light is one of the main environmental cues that affects the physiology and behavior of many organisms. The effect of light on genome-wide transcriptional regulation has been well-studied in green algae and plants, but not in red algae. Cyanidioschyzon merolae is used as a model red algae, and is suitable for studies on transcriptomics because of its compact genome with a relatively small number of genes. In addition, complete genome sequences of the nucleus, mitochondrion, and chloroplast of this organism have been determined. Together, these attributes make C. merolae an ideal model organism to study the response to light stimuli at the transcriptional and the systems biology levels. Previous studies have shown that light significantly affects cell signaling in this organism, but there are no reports on its blue light- and red light-mediated transcriptional responses. We investigated the direct effects of blue and red light at the transcriptional level using RNA-seq. Blue and red lights were found to regulate 35 % of the total genes in C. merolae. Blue light affected the transcription of genes involved in protein synthesis while red light specifically regulated the transcription of genes involved in photosynthesis and DNA repair. Blue or red light regulated genes involved in carbon metabolism and pigment biosynthesis. Overall, our data showed that red and blue light regulate the majority of the cellular, cell division, and repair processes in C. merolae.

Development of a transposon-based marker system for mutation breeding in sorghum (Sorghum bicolor L.).

Under certain circumstances, transposable elements (TE) can create or reverse mutations and alter the genome size of a cell. Sorghum (Sorghum bicolor L.) is promising for plant transposon tagging due to its small genome size and its low content of repetitive DNA. We developed a marker system based on targeted region amplification polymorphisms (TE-TRAP) that uses the terminal inverted repeats (TIRs) of transposons. A total of 3816 class 2 transposons belonging to the PIF/Harbinger family were identified from the whole sorghum genome that produced five primers, including eight types of TIRs. To define the applicability and utilization of TE-TRAP, we used 21 individuals that had been bred after ɤ-ray irradiation. In total, 31 TE-TRAP, 16 TD, and 21 AFLP primer combinations generated 1133, 223, and 555 amplicons, respectively. The percent polymorphic marker was 62.8, 51.1, and 59.3% for the TE-TRAP, TD, and AFLP markers, respectively. Phylogenetic and principal component analyses revealed that TE-TRAP divided the 21 individuals into three groups. Analysis of molecular variance suggested that TE-TRAP had a higher level of genetic diversity than the other two marker systems. After verifying the efficiency of TE-TRAP, 189 sorghum individuals were used to investigate the associations between the markers and the ɤ-ray doses. Two significant associations were found among the polymorphic markers. This TE-based method provides a useful marker resource for mutation breeding research.

The conserved factor DE-ETIOLATED 1 cooperates with CUL4-DDB1DDB2 to maintain genome integrity upon UV stress.

Plants and many other eukaryotes can make use of two major pathways to cope with mutagenic effects of light, photoreactivation and nucleotide excision repair (NER). While photoreactivation allows direct repair by photolyase enzymes using light energy, NER requires a stepwise mechanism with several protein complexes acting at the levels of lesion detection, DNA incision and resynthesis. Here we investigated the involvement in NER of DE-ETIOLATED 1 (DET1), an evolutionarily conserved factor that associates with components of the ubiquitylation machinery in plants and mammals and acts as a negative repressor of light-driven photomorphogenic development in Arabidopsis. Evidence is provided that plant DET1 acts with CULLIN4-based ubiquitin E3 ligase, and that appropriate dosage of DET1 protein is necessary for efficient removal of UV photoproducts through the NER pathway. Moreover, DET1 is required for CULLIN4-dependent targeted degradation of the UV-lesion recognition factor DDB2. Finally, DET1 protein is degraded concomitantly with DDB2 upon UV irradiation in a CUL4-dependent mechanism. Altogether, these data suggest that DET1 and DDB2 cooperate during the excision repair process.

Evidence for a role of Arabidopsis CDT1 proteins in gametophyte development and maintenance of genome integrity.

Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase ε, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability.

Genome-wide mapping of the HY5-mediated gene networks in Arabidopsis that involve both transcriptional and post-transcriptional regulation.

LONG HYPOCOTYL 5 (HY5) is a basic leucine zipper transcription factor (TF) that functions downstream of multiple families of photoreceptors. Mutations in the HY5 gene cause a myriad of aberrant phenotypes in Arabidopsis, including elongated hypocotyl, reduced accumulation of pigments, halted chloroplast development in greening hypocotyls, altered root morphology, and defective hormonal and stimulus responses. HY5 thus acts as an integrator that links various gene networks to coordinate plant development. Here we report the experimental mapping of HY5-mediated gene networks in Arabidopsis by integrating genomic loci occupied by HY5 and HY5-dependent gene expression profiles. Our results indicate that HY5 binds to over 9000 genes, detectably affecting the expression of over 1100 genes, either positively or negatively. Further, HY5 indirectly regulate many other genes through sub-networks mediated by other regulators. In particular, HY5 regulates eight miRNA genes that in turn control the transcript abundance of specific target genes. Over-expressing HY5-targeted miR408 resulted in phenotypes that are opposite to the hy5 mutants. Together, our results reveal both transcriptional and post-transcriptional components of the HY5-mediated gene networks.

The point mutation induced by the low-energy N+ ion implantation in impatiens balsamine genome.

To investigate the effect of genome mutations induced by low energy ions implantation in higher plants, genome mutation of Impatiens balsamine mutant induced by low energy N+ ion implantation were analyzed by the RAPD, ISSR and genome sequence. Six out of the 121 ISSR primers and 6 out of the 135 RAPD primers showed that polymorphism ratios between mutants and wild type were 4.96% and 2.89%, respectively. Sequence analysis revealed that base deletions, insertions, and substitutions were observed in the mutant genome comparable to wild type. N+ induced point mutations were mostly base substitution (77.4%), no duplication, long fragments insertions and deletions was found. In all point mutation, adenine (A) was most sensitive to the N+ ion implantation in impatiens. The transition was mainly A --> guanine (G) (15.90%) and thymine (T) --> cytosine (C) (12.55%). Transversion happened in A <--> T (16.74%), which much higher than C <--> G(5.02%), G <--> T(6.69%), A <--> C (7.11%) bases. These findings indicate that low energy ions being a useful mutagen were mostly cause the point mutation in impatiens.

Regulation and role of Arabidopsis CUL4-DDB1A-DDB2 in maintaining genome integrity upon UV stress.

Plants use the energy in sunlight for photosynthesis, but as a consequence are exposed to the toxic effect of UV radiation especially on DNA. The UV-induced lesions on DNA affect both transcription and replication and can also have mutagenic consequences. Here we investigated the regulation and the function of the recently described CUL4-DDB1-DDB2 E3 ligase in the maintenance of genome integrity upon UV-stress using the model plant Arabidopsis. Physiological, biochemical, and genetic evidences indicate that this protein complex is involved in global genome repair (GGR) of UV-induced DNA lesions. Moreover, we provide evidences for crosstalks between GGR, the plant-specific photo reactivation pathway and the RAD1-RAD10 endonucleases upon UV exposure. Finally, we report that DDB2 degradation upon UV stress depends not only on CUL4, but also on the checkpoint protein kinase Ataxia telangiectasia and Rad3-related (ATR). Interestingly, we found that DDB1A shuttles from the cytoplasm to the nucleus in an ATR-dependent manner, highlighting an upstream level of control and a novel mechanism of regulation of this E3 ligase.

Identification and characterization of inheritable structural variations induced by ion beam radiations in rice.

High energy ion beams are effective physical mutagens for mutation induction in plants. Due to their high linear energy transfer (LET) property, they are known to generate single nucleotide variations (SNVs) and insertion/deletions (InDels, <50 bp) as well as structural variations (SVs). However, due to the technical difficulties to identify SVs, studies on ion beam induced SVs by genome sequencing have so far been limited in numbers and inadequate in nature, and knowledge of SVs is scarce with regards to their characteristics. In the present study, we identified and validated SVs in six M4 plants (designated as Ar_50, Ar_100, C_150, C_200, Ne_50 and Ne_100 according to ion beam types and irradiation doses), two each induced by argon (40Ar18+), carbon (12C6+) and neon (20Ne10+) ion beams and performed in depth analyses of their characteristics. In total, 22 SVs were identified and validated, consisting of 11 deletions, 1 duplication, and 4 intra-chromosomal and 6 inter-chromosomal translocations. There were several SVs larger than 1 kbp. The SVs were distributed across the whole genome with an aggregation with SNVs and InDels only in the Ne_50 mutants. An enrichment of a 11-bp wide G-rich DNA motif 'GAAGGWGGRGG' was identified around the SV breakpoints. Three mechanisms might be involved in the SV formation, i.e., the expansion of tandem repeats, transposable element insertion, and non-allelic homologous recombination. Put together, the present study provides a preliminary view of SVs induced by Ar, C and Ne ion beam radiations, and as a pilot study, it contributes to our understanding of how SVs might form after ion beam irradiation in rice.

Comparison and Characterization of Mutations Induced by Gamma-Ray and Carbon-Ion Irradiation in Rice (Oryza sativa L.) Using Whole-Genome Resequencing.

Gamma-rays are the most widely used mutagenic radiation in plant mutation breeding, but detailed characteristics of mutated DNA sequences have not been clarified sufficiently. In contrast, newly introduced physical mutagens, e.g., heavy-ion beams, have attracted geneticists' and breeders' interest and many studies on their mutation efficiency and mutated DNA characteristics have been conducted. In this study, we characterized mutations induced by gamma rays and carbon(C)-ion beams in rice (Oryza sativa L.) mutant lines at M5 generation using whole-genome resequencing. On average, 57.0 single base substitutions (SBS), 17.7 deletions, and 5.9 insertions were detected in each gamma-ray-irradiated mutant, whereas 43.7 single SBS, 13.6 deletions, and 5.3 insertions were detected in each C-ion-irradiated mutant. The structural variation (SV) analysis detected 2.0 SVs (including large deletions or insertions, inversions, duplications, and reciprocal translocations) on average in each C-ion-irradiated mutant, while 0.6 SVs were detected on average in each gamma-ray-irradiated mutant. Furthermore, complex SVs presumably having at least two double-strand breaks (DSBs) were detected only in C-ion-irradiated mutants. In summary, gamma-ray irradiation tended to induce larger numbers of small mutations than C-ion irradiation, whereas complex SVs were considered to be the specific characteristics of the mutations induced by C-ion irradiation, which may be due to their different radiation properties. These results could contribute to the application of radiation mutagenesis to plant mutation breeding.

Characterization of hemizygous deletions in citrus using array-comparative genomic hybridization and microsynteny comparisons with the poplar genome.

BACKGROUND: Many fruit-tree species, including relevant Citrus spp varieties exhibit a reproductive biology that impairs breeding and strongly constrains genetic improvements. In citrus, juvenility increases the generation time while sexual sterility, inbreeding depression and self-incompatibility prevent the production of homozygous cultivars. Genomic technology may provide citrus researchers with a new set of tools to address these various restrictions. In this work, we report a valuable genomics-based protocol for the structural analysis of deletion mutations on an heterozygous background. RESULTS: Two independent fast neutron mutants of self-incompatible clementine (Citrus clementina Hort. Ex Tan. cv. Clemenules) were the subject of the study. Both mutants, named 39B3 and 39E7, were expected to carry DNA deletions in hemizygous dosage. Array-based Comparative Genomic Hybridization (array-CGH) using a Citrus cDNA microarray allowed the identification of underrepresented genes in these two mutants. Subsequent comparison of citrus deleted genes with annotated plant genomes, especially poplar, made possible to predict the presence of a large deletion in 39B3 of about 700 kb and at least two deletions of approximately 100 and 500 kb in 39E7. The deletion in 39B3 was further characterized by PCR on available Citrus BACs, which helped us to build a partial physical map of the deletion. Among the deleted genes, ClpC-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll b synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll a/b ratio in green tissues. CONCLUSION: In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on Citrus clementina. The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with Populus trichocarpa than with the phylogenetically closer Arabidopsis thaliana. This work corroborates the potential of Citrus genomic resources to assist mutagenesis-based approaches for functional genetics, structural studies and comparative genomics, and hence to facilitate citrus variety improvement.

Radiation induced alterations in Vigna radiata during in vitro somatic embryogenesis.

PURPOSE: Tissue culture has been exploited to understand molecular aspects of regeneration potential of the plants in normal and in stressed conditions. The present study describes ionizing radiation from (60)Co source as the stress stimulator to assess in vitro development of somatic embryo of Vigna radiata, a protein-rich pulse. MATERIALS AND METHODS: Callus culture was established, using leaves of V. radiata. Somatic embryogenesis was induced by manipulating plant hormones. Calli were exposed to gamma rays. Genomic DNA isolated from gamma-irradiated callus samples were subjected to random amplified polymorphic DNA analysis. A band of molecular weight 1440 bp was used as a probe and Southern hybridization was carried out. To determine alterations in DNA following irradiation, RAPD bands were cloned and sequenced from control and irradiated samples. Embryogenic calli were exposed to gamma irradiation and the effects were assessed immediately and after seven days of exposure. Phenotypic alterations were observed using scanning electron microscopy. RESULTS: Exposed calli revealed altered frequency of somatic embryo formation. Results showed that the 1440 bp molecular weight probe hybridized with bands of low molecular weight. DNA sequences from irradiated samples showed recombination when compared to control. Scanning electron micrography illustrated presence of transient pores on the exposed embryos. BLAST search of the DNA sequences showed partial homology with some sequences from Arabidopsis thaliana. CONCLUSION: The present report might help in designing a breeding program, where both radiation coupled with somatic embryogenesis could be employed to build up the desired variants.

Genome-wide excision repair in Arabidopsis is coupled to transcription and reflects circadian gene expression patterns.

Plants are exposed to numerous DNA-damaging stresses including the exposure to ultraviolet (UV) component of solar radiation. They employ nucleotide excision repair to remove DNA-bulky adducts and to help eliminate UV-induced DNA lesions, so as to maintain their genome integrity and their fitness. Here, we generated genome-wide single-nucleotide resolution excision repair maps of UV-induced DNA damage in Arabidopsis at different circadian time points. Our data show that the repair of UV lesions for a large fraction of the genome is controlled by the joint actions of the circadian clock and transcription by RNA polymerase II. Our findings reveal very strong repair preference for the transcribed strands of active genes in Arabidopsis, and 10-30% of the transcription-coupled repair is circadian time-dependent. This dynamic range in nucleotide excision repair levels throughout the day enables Arabidopsis to cope with the bulky DNA lesion-inducing environmental factors including UV.

Low-energy ion beam promotes the transcription and transposition of the Copia-retrotransposons in wheat (Triticum aestivum L.).

LTR-retrotransposons are genetic elements having the direct long terminal repeats (LTRs). It can move via an RNA intermediate within genomes and is an important fraction of eukaryote genomes. Low-energy N(+) ion beam promoted the transcription of the copia-retransposons in those wheat (cv. 'Zhoumai 16', which were radiated and allowed to grow for 24 h and 48 h from the planting. Relative expression ratio of the copia-retransposons was elevated in different degrees (with a max 40 fold) in wheat plants treated with different doses of N(+) beam, comparing to that in the controls. The molecule markers of the IRAP and REMAP to the DNA isolated from the 14-d leaves of wheat plants treated with the low-energy N(+) beam showed that the transposition of some copia-retransposons was re-activated. The enhanced transcription of the copia-retransposons in wheat could weaken or enhance the expression of their nearby genes. The transposition of the retrotransposon in genome can change the primary structure of the functional DNA fragments of chromosomes, and it can also be visualized as the appearance of a new phenotype of plants. In the mid 1980s, the biological effects of low-energy ion beam were recognized and demonstrated experimentally. Hence, it suggests that the enhanced transcription and the re-activated transposition of the retrotransposons are partially attributed to the biological effect of low-energy ion beam.

Acute exposure to UVB has a more profound effect on plant genome stability than chronic exposure.

Environmental factors that damage DNA have various lengths of exposure and intensity levels. Although the results of increasing the intensity of a DNA damaging agent is often predictable, it is not clear whether the stage during development when the exposure is received has any influence on the amount of DNA damage. In this paper we analyzed the influence of UVB on the stability of Arabidopsis thaliana and the Nicotiana tabacum genomes. Our experiments showed that the acute exposure to UVB produces a significantly greater increase in homologous recombination frequency (HRF) and recombination rate (RR) compared with that produced by chronic exposure. The increase in HRF showed a positive correlation with UVB dose and a negative correlation with plant age. In other words, as the UVB dose was increased, there was a concomitant increase in HRF. Conversely, older plants had a lower HRF increase as compared to younger plants. Our experiments suggest that exposure to UVB makes the most significant impact on genome stability during the early stages of plant development.

Genome-wide analysis of radiation-induced mutations in rice (Oryza sativa L. ssp. indica).

Radiation has been efficiently used for rice germplasm innovation. However, the molecular mechanisms by which radiation induces mutations are still unclear. In this study, we performed whole genome sequencing to reveal the comprehensive mutations in rice treated with radiation. Red-1 (a rice rich in beneficial ingredients for human health) was derived from rice 9311 after γ-radiation. Solexa sequencing technology was applied to uncover the mutations. Compared with the 9311 genome, 9.19% of genome sequences were altered in the Red-1 genome. Among these alterations, there were 381,403 SNPs, 50,116 1-5 bp Indels, 1279 copy number variations, and 10,026 presence/absence variations. These alterations were located in 14,493 genes, the majority of which contained a kinase domain, leucine rich repeats, or Cyt_P450. Point mutations were the main type of variation in the Red-1 genome. Gene ontology clustering revealed that genes that are associated with cell components, binding function, catalytic activity and metabolic processes were susceptible to γ-radiation. It was also predicted that 8 mutated genes were involved in the biosynthetic pathways of beneficial products or pigment accumulation. We conclude that genome-wide analysis of mutations provides novel insights into the mechanisms by which radiation improves the beneficial ingredients in rice Red-1.

Construction of whole genome radiation hybrid panels and map of chromosome 5A of wheat using asymmetric somatic hybridization.

To explore the feasibility of constructing a whole genome radiation hybrid (WGRH) map in plant species with large genomes, asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Bupleurum scorzonerifolium Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV) and gamma-ray and rescued by protoplast fusion using B. scorzonerifolium as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII) that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ∼501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.

Kinetic transcriptomic approach revealed metabolic pathways and genotoxic-related changes implied in the Arabidopsis response to ionising radiations.

Plants exposed to ionising radiation (IR) have to face direct and indirect (oxidative stress) deleterious effects whose intensity depends on the dose applied and led to differential genome regulation. Transcriptomic analyses were conducted with CATMA microarray technology on Arabidopsis thaliana plantlets, 2 and 26h after exposure to the IR doses 10Gy and 40Gy. 10Gy treatment seemed to enhance antioxidative compound biosynthetic pathways whereas the 40Gy dose up-regulated ROS-scavenging enzyme genes. Transcriptomic data also highlighted a differential regulation of chloroplast constituent genes depending on the IR dose, 10Gy stimulating and 40Gy down-regulating. This probable 40Gy decrease of photosynthesis could help for the limitation of ROS production and may be coupled with programmed cell death (PCD)/senescence phenomena. Comparisons with previous transcriptomic studies on plants exposed to a 100Gy dose revealed 60 dose-dependent up-regulated genes, including notably cell cycle checkpoints to allow DNA repairing phenomena. Furthermore, the alteration of some cellular structure related gene expression corroborated a probable mitotic arrest after 40Gy. Finally, numerous heat-shock protein and chaperonin genes, known to protect proteins against stress-dependent dysfunction, were up-regulated after IR exposure.