SUMMARYDNA repair is important for maintaining genome integrity. In plants, DNA damage accumulated in the embryo of seeds is repaired early in imbibition, and is important for germination performance and seed longevity. An essential step in most repair pathways is the DNA ligase-mediated rejoining of single-and double-strand breaks. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Here, we report the characterization of DNA LIGASE VI, which is only found in plant species. The primary structure of this ligase shows a unique N-terminal region that contains a b-CASP motif, which is found in a number of repair proteins, including the DNA double-strand break (DSB) repair factor Artemis. Phenotypic analysis revealed a delay in the germination of atlig6 mutants compared with wild-type lines, and this delay becomes markedly exacerbated in the presence of the genotoxin menadione. Arabidopsis atlig6 and atlig6 atlig4 mutants display significant hypersensitivity to controlled seed ageing, resulting in delayed germination and reduced seed viability relative to wild-type lines. In addition, atlig6 and atlig6 atlig4 mutants display increased sensitivity to low-temperature stress, resulting in delayed germination and reduced seedling vigour upon transfer to standard growth conditions. Seeds display a rapid transcriptional DNA DSB response, which is activated in the earliest stages of water imbibition, providing evidence for the accumulation of cytotoxic DSBs in the quiescent seed. These results implicate AtLIG6 and AtLIG4 as major determinants of Arabidopsis seed quality and longevity.
SummaryDNA damage threatens the integrity of the genome and has potentially lethal consequences for the organism. Plant DNA is under continuous assault from endogenous and environmental factors and effective detection and repair of DNA damage are essential to ensure the stability of the genome. One of the most cytotoxic forms of DNA damage are DNA double-strand breaks (DSBs) which fragment chromosomes. Failure to repair DSBs results in loss of large amounts of genetic information which, following cell division, severely compromises daughter cells that receive fragmented chromosomes. This review will survey recent advances in our understanding of plant responses to chromosomal breaks, including the sources of DNA damage, the detection and signalling of DSBs, mechanisms of DSB repair, the role of chromatin structure in repair, DNA damage signalling and the link between plant recombination pathways and transgene integration. These mechanisms are of critical importance for maintenance of plant genome stability and integrity under stress conditions and provide potential targets for the improvement of crop plants both for stress resistance and for increased precision in the generation of genetically improved varieties.
Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production.DNA repair | seed vigor | DNA damage response | dormancy cycling | soil seed bank M aintenance of genome integrity is indispensable for cellular survival and transmission of genetic information to the next generation; however, constant exposure of DNA to environmental and cellular oxidative stresses results in damage that can arrest growth and result in mutagenesis or cell death. Consequently, organisms have evolved powerful DNA repair and DNA damage signaling mechanisms. In plants, as in other eukaryotes, the cellular response to DNA damage is orchestrated by the phosphoinositide-3-kinase-related protein kinases (PIKKs) ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR) (1). In response to genotoxic stresses, these checkpoint kinases activate DNA repair factors, delay or halt cell cycle progression, and promote endocycles or programmed cell death (1-4). ATM is activated by double-strand DNA breaks (DSBs), a highly toxic form of DNA damage that results in chromosome fragmentation (1, 5), and plants mutated in ATM display hypersensitivity to DSBs induced by gamma radiation or radiomimetics (6). ATM also mediates a strong transcriptional up-regulation of hundreds of genes, including the SIAMESE/SIAMESE-RELATED cell cycle inhibitors ...
Seeds are important to agriculture and conservation of plant biodiversity. In agriculture, seed germination performance is an important determinant of crop yield, in particular under adverse climatic conditions. Deterioration in seed quality is associated with the accumulation of cellular damage to macromolecules including lipids, protein, and DNA. Mechanisms that mitigate the deleterious cellular damage incurred in the quiescent state and in cycles of desiccation-hydration are crucial for the maintenance of seed viability and germination vigour. In early-imbibing seeds, damage to the embryo genome must be repaired prior to initiation of cell division to minimize growth inhibition and mutation of genetic information. Here we review recent advances that have established molecular links between genome integrity and seed quality. These studies identified that maintenance of genome integrity is particularly important to the seed stage of the plant lifecycle, revealing new insight into the physiological roles of plant DNA repair and recombination mechanisms. The high conservation of DNA repair and recombination factors across plant species underlines their potential as promising targets for the improvement of crop performance and development of molecular markers for prediction of seed vigour.
SummaryThe ability of plants to repair DNA double-strand breaks (DSBs) is essential for growth and fertility. The Arabidopsis DSB repair proteins AtRAD50 and AtMRE11 form part of an evolutionarily conserved complex that, in Saccharomyces cerevisiae and mammals, includes a third component termed XRS2 and NBS1, respectively. The MRN complex (MRX in yeast) has a direct role in DSB repair and is also required for DNA damage signaling and checkpoint activation in a pathway mediated by the protein kinase ATM. This study characterizes Arabidopsis and maize NBS1 orthologues that share conserved protein motifs with human NBS1. Both plant NBS1 proteins interact with the corresponding MRE11 orthologues, and deletion analysis of AtNBS1 defines a region towards the C-terminus (amino acids 465-500) that is required for interaction with AtMRE11. Arabidopsis lines homozygous for a T-DNA insertional mutation in AtNBS1 display hypersensitivity to the DNA cross-linking reagent mitomycin C, and this phenotype can be rescued by complementation with the wild-type gene, consistent with a function for AtNBS1 in plant DSB repair. Analysis of atnbs1-1 atatm double mutants revealed a role for AtNBS1 in meiotic recombination. While atatm mutants produce reduced seed numbers, plants deficient in both AtATM and AtNBS1 are completely infertile. Cytological analysis of these double mutants revealed incomplete chromosome pairing and synapsis in meiotic prophase, and extensive chromosome fragmentation in metaphase I and subsequent stages. These results suggest a novel role for AtNBS1 that is independent of AtATM-mediated signaling and functions in the very early stages of meiosis.
Successful germination represents a crucial developmental transition in the plant lifecycle and is important both for crop yields and plant survival in natural ecosystems. However, germination potential decreases during storage and seed longevity is a key determinant of crop production. Decline in germination vigor is initially manifest as an increasing delay to radicle emergence and the completion of germination and eventually culminating in loss of seed viability. The molecular mechanisms that determine seed germination vigor and viability remain obscure, although deterioration in seed quality is associated with the accumulation of damage to cellular structures and macromolecules including lipids, protein, and nucleic acids. In desiccation tolerant seeds, desiccation/rehydration cycles and prolonged periods in the dry quiescent state are associated with remarkable levels of stress to the embryo genome which can result in mutagenesis of the genetic material, inhibition of transcription and replication and delayed growth and development. An increasing number of studies are revealing DNA damage accumulated in the embryo genome, and the repair capacity of the seed to reverse this damage, as major factors that determine seed vigor and viability. Recent findings are now establishing important roles for the DNA damage response in regulating germination, imposing a delay to germination in aged seed to minimize the deleterious consequences of DNA damage accumulated in the dry quiescent state. Understanding the mechanistic basis of seed longevity will underpin the directed improvement of crop varieties and support preservation of plant genetic resources in seed banks.
SummaryDouble-strand breaks (DSBs) in DNA may occur spontaneously in the cell or be induced experimentally by g-irradiation, and represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination appears to be the major pathway for DSB repair in humans and plants, and it may also be the major route whereby T-DNA integrates into the plant genome during cell transformation. In yeast and mammals, the exposed ends of damaged DNA are bound with high af®nity by a dimer of Ku70 and Ku80 proteins, which protects the ends from exonucleases and juxtaposes the two ends of the DSB, independent of sequence homology. Here we report the functional characterization of Ku70 and Ku80 from Arabidopsis thaliana, and demonstrate that AtKu80 and AtKu70 form a heterodimer with DNA binding activity that is speci®c for DNA ends. An atku80 knockout mutant shows hypersensitivity to the DNA-damaging agents menadione and bleomycin, consistent with a role for AtKu80 in the repair of DSBs in vivo in Arabidopsis.
DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.
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