Highlights • Hef may function in stalled fork repair by association of its IDR with multiple partners. • EndoQ cleaves the phosphodiester bond 5′ from dI, dU, dX, and the AP site in DNA strand. • ...PprA plays a principal role in CNDEJ by forming a larger nucleoprotein complex. • PprI cleaves DdrO, a radiation-inducible repressor of RDR regulon.
Deinococcus spp. are known for their radiation resistance, toxic compound removal, and production of valuable substances. Therefore, developing gene expression systems for Deinococcus spp. is crucial ...in advancing genetic engineering applications. To date, plasmid vectors that express foreign genes in D. radiodurans and D. geothermalis have been limited to plasmid pI3 and its derivatives. In contrast, plasmid vectors that express foreign genes in D. grandis include plasmid pZT23 and its derivatives. In this study, we developed a new system for the stable introduction and retention of expression plasmids for D. grandis . Two cryptic plasmids were removed from the wild-type strain to generate the TY3 strain. We then constructed a shuttle vector plasmid, pGRC5, containing the replication initiation region of the smallest cryptic plasmid, pDEGR-3, replication initiation region of the E. coli vector, pACYC184, and an antibiotic resistance gene. We introduced pGRC5, pZT23-derived plasmid pZT29H, and pI3-derived plasmid pRADN8 into strain TY3, and found their coexistence in D. grandis cells. The quantitative PCR assay results found that pGRC5, pZT29H, and pRADN8 had relative copy numbers of 11, 26, and 5 per genome, respectively. Furthermore, we developed a new plasmid in which the luciferase gene was controlled by the promoter region, which contained radiation-desiccation response operator sequences for D. grandis DdrO, a stress response regulon repressor in D. grandis , hence inducing gene expression via ultraviolet-C light irradiation. These plasmids are expected to facilitate the removal and production of toxic and valuable substances, in D. grandis , respectively, particularly of those involving multiple genes.
Abstract
Ionizing radiation induces genetic variations in plants, which makes it useful for plant breeding. A theory that the induced mutations occur randomly in the genome has long been accepted, ...but is now controversial. Nevertheless, a comparative analysis of the mutations at multiple loci has not been conducted using irradiated M
1
genomes that contain all types of mutations. In this study, we identified Arabidopsis mutants (
pab2
and
pab3
) in a mutagenized population of an anthocyanin-positive seed mutant (
ban
). Both
pab2
and
pab3
were revealed to be double mutants (
tt4 ban
and
tt8 ban
, respectively) that produced similar anthocyanin-less immature seeds, but differentially colored mature seeds. These features enabled the seed color-based detection of de novo M
1
mutations in
TT4
or
TT8
following the irradiation of double heterozygous plants (
TT4
/
tt4 TT8
/
tt8 ban
/
ban
). Most of the irradiated double heterozygous plants produced anthocyanin-positive immature seeds, but 19 plants produced anthocyanin-less immature seeds. Of these 19 mutants, 2 and 17 exhibited
tt4
- and
tt8
-type mature seed coloration, respectively. The molecular analysis of the seed coat DNA from randomly selected anthocyanin-less seeds detected mutations at the locus predicted on the basis of the phenotype. Thus, the simple system developed in this study can reliably detect radiation-induced mutations at multiple loci in irradiated Arabidopsis M
1
plants.
The hypothesis called “panspermia” proposes an interplanetary transfer of life. Experiments have exposed extremophilic organisms to outer space to test microbe survivability and the panspermia ...hypothesis. Microbes inside shielding material with sufficient thickness to protect them from UV-irradiation can survive in space. This process has been called “lithopanspermia,” meaning rocky panspermia. We previously proposed sub-millimeter cell pellets (aggregates) could survive in the harsh space environment based on an on-ground laboratory experiment. To test our hypothesis, we placed dried cell pellets of the radioresistant bacteria
Deinococcus
spp. in aluminum plate wells in exposure panels attached to the outside of the International Space Station (ISS). We exposed microbial cell pellets with different thickness to space environments. The results indicated the importance of the aggregated form of cells for surviving in harsh space environment. We also analyzed the samples exposed to space from 1 to 3 years. The experimental design enabled us to get and extrapolate the survival time course to predict the survival time of
Deinococcus radiodurans
. Dried deinococcal cell pellets of 500 μm thickness were alive after 3 years of space exposure and repaired DNA damage at cultivation. Thus, cell pellets 1 mm in diameter have sufficient protection from UV and are estimated to endure the space environment for 2–8 years, extrapolating the survival curve and considering the illumination efficiency of the space experiment. Comparison of the survival of different DNA repair-deficient mutants suggested that cell aggregates exposed in space for 3 years suffered DNA damage, which is most efficiently repaired by the
uvrA
gene and
uvdE
gene products, which are responsible for nucleotide excision repair and UV-damage excision repair. Collectively, these results support the possibility of microbial cell aggregates (pellets) as an ark for interplanetary transfer of microbes within several years.
Size of mutant sector and genetic damage were evaluated in Arabidopsis to further our understanding of effective ion beam use in plant mutation breeding. Arabidopsis seeds, heterozygous for the ...GLABRA1 (GL1) gene (GL1/gl1-1), were irradiated with 15.8MeV/u neon ions (mean linear energy transfer (LET): 352keV/μm), 17.3MeV/u carbon ions (113keV/μm), or 60Co gamma rays. The frequency and size of glabrous sectors generated because of inactivation of the GL1 allele were examined. The frequency and overall size of large deletions were evaluated based on the loss of heterozygosity of DNA markers using DNA isolated from glabrous tissue. Irrespective of the radiation properties, plants with mutant sectors were obtained at similar frequencies at the same effective dosage necessary for survival reduction. Ion beams tended to induce larger mutant sectors than gamma rays. The frequency of large deletions (>several kbp) increased as the LET value increased, with chromosome regions larger than 100kbp lost in most large deletions. The distorted segregation ratio of glabrous plants in the progenies of irradiated GL1/gl1-1 plants suggested frequent occurrence of chromosome rearrangement, especially those subjected to neon ions. Exposure to ion beams with moderate LET values (30–110keV/μm) is thought effective for inducing mutant sectors without causing extensive genetic damage.
To gain insight into the mutagenic effects of accelerated heavy ions in plants, the mutagenic effects of carbon ions near the range end (mean linear energy transfer (LET): 425keV/μm) were compared ...with the effects of carbon ions penetrating the seeds (mean LET: 113keV/μm). Mutational analysis by plasmid rescue of Escherichia coli rpsL from irradiated Arabidopsis plants showed a 2.7-fold increase in mutant frequency for 113keV/μm carbon ions, whereas no enhancement of mutant frequency was observed for carbon ions near the range end. This suggested that carbon ions near the range end induced mutations that were not recovered by plasmid rescue. An Arabidopsis DNA ligase IV mutant, deficient in non-homologous end-joining repair, showed hyper-sensitivity to both types of carbon-ion irradiation. The difference in radiation sensitivity between the wild type and the repair-deficient mutant was greatly diminished for carbon ions near the range end, suggesting that these ions induce irreparable DNA damage. Mutational analysis of the Arabidopsis GL1 locus showed that while the frequency of generation of glabrous mutant sectors was not different between the two types of carbon-ion irradiation, large deletions (>∼30kb) were six times more frequently induced by carbon ions near the range end. When 352keV/μm neon ions were used, these showed a 6.4 times increase in the frequency of induced large deletions compared with the 113keV/μm carbon ions. We suggest that the proportion of large deletions increases with LET in plants, as has been reported for mammalian cells. The nature of mutations induced in plants by carbon ions near the range end is discussed in relation to mutation detection by plasmid rescue and transmissibility to progeny.
Arabidopsis TRANSPARENT TESTA19 (TT19) encodes a glutathione-S-transferase (GST)-like protein that is involved in the accumulation of proanthocyanidins (PAs) in the seed coat. PA accumulation sites ...in tt19 immature seeds were observed as small vacuolar-like structures, whereas those in tt12, a mutant of the tonoplast-bound transporter of PAs, and tt12 tt19 were observed at peripheral regions of small vacuoles. We found that tt19 immature seeds had small spherical structures showing unique thick morphology by differential interference contrast microscopy. The distribution pattern of the thick structures overlapped the location of PA accumulation sites, and the thick structures were outlined with GFP-TT12 proteins in tt19. PA analysis showed higher (eightfold) levels of solvent-insoluble PAs in tt19 immature seeds compared with the wild type. Metabolic profiling of the solvent-soluble fraction by LC-MS demonstrated that PA derivatives such as epicatechins and epicatechin oligomers, although highly accumulated in the wild type, were absent in tt19. We also revealed that tt12 specifically accumulated glycosylated epicatechins, the putative transport substrates for TT12. tt12 tt19 showed a similar metabolic profile to tt19. Given the cytosolic localization of functional GFP-TT19 proteins, our results suggest that TT19, which acts prior to TT12, functions in the cytosol to maintain the regular accumulation of PA precursors, such as epicatechin and glycosylated epicatechin, in the vacuole. The PA pathway in the Arabidopsis seed coat is discussed in relation to the subcellular localization of PA metabolites.
Thermococcus kodakarensis possesses two DNA polymerases, Pol B and Pol D. We generated a T. kodakarensis strain (DPB1) in which polB was completely deleted and a derivative of DPB1 in which polB was ...overexpressed; neither of the generated strains exhibited any growth delay, indicating that the lack or overexpression of Pol B in T. kodakarensis did not affect cell growth. We also found that DPB1 showed higher sensitivity to four DNA-damaging agents (ultraviolet C irradiation, γ-ray irradiation, methyl methanesulfonate, and mitomycin C) than the parental strain. The sensitivity of DPB1 was restored to the level of the parent strain by the introduction of a plasmid harboring polB, suggesting that the DNA damage-sensitive phenotype of DPB1 was due to the loss of polB. Collectively, these results indicate that Pol B is involved in DNA repair, but not DNA replication, which, in turn, implies that Pol D is the sole replicative DNA polymerase in Thermococcus species.
Recent transcriptome analysis revealed that
Deinococcus radiodurans efficiently coordinate their recovery from ionizing radiation through a complex network of DNA repair and metabolic pathway ...switching. However, the additional discovery of numerous irradiation-response genes has provided new targets for the identification of genes primarily crucial to radiation resistance. Investigations based on electron microscopy suggest that the observed radiation resistance in
D. radiodurans might be partly caused by the presence of an unusual ring-like conformation of nucleoids. Although such investigations provide useful insights into the mechanisms underlying radiation resistance, a more detailed empirical explanation of why
D. radiodurans is so radiation resistant is still needed. Further research based on alternative genetic and biochemical approaches should help to gain a better understanding of the mechanisms involved in DNA repair.
Some Rumex species such as sorrel are edible as baby leaf salad greens. On the other hand, Rumex plants accumulate soluble oxalate, a toxic metabolite which causes serious diseases such as renal ...syndrome. We attempted to produce low-oxalate plants of R. obtusifolius, a perennial weed which has higher vitamin C and amino acid content and higher tolerance to stress than many other Rumex species. Ion beams are ionising radiation with high linear energy transfer that causes a wide spectrum of mutations. Thus, in the present study we evaluated the effects of ion beams on oxalate and other primary metabolites in leaves of R. obtusifolius using CE-MS. The results showed that oxalate content was increased by irradiation with carbon ion beams. Metabolome analysis revealed that ion beams affected carbon flow to the isocitrate pathway, which is involved in oxalate synthesis. These observations suggested that modulation of carbon flow to the isocitrate pathway is important to regulate oxalate levels in plants.
•The ion beam-irradiation on seeds enhanced oxalate accumulation in leaves.•Precursors of oxalates were also increased by the ion beam-irradiation.•Contents of several amino acids were decreased depend on oxalate accumulation.
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