RESUMEN
The WHIRLY (WHY) family of DNA/RNA binding proteins fulfil multiple but poorly characterised functions in plants. We analysed WHY protein functions in the Arabidopsis Atwhy1, Atwhy3, Atwhy1why3 single and double mutants and wild type controls. The Atwhy3 and Atwhy1why3 double mutants showed a significant delay in flowering, having more siliques per plant but with fewer seeds per silique than the wild type. While germination was similar in the unaged high-quality seeds of all lines, significant decreases in vigour and viability were observed in the aged mutant seeds compared with the wild type. Imbibition of unaged high-quality seeds was characterised by large increases in transcripts that encode proteins involved in oxygen sensing and responses to hypoxia. Seed aging resulted in a disruption of the imbibition-induced transcriptome profile such that transcripts encoding RNA metabolism and processing became the most abundant components of the imbibition signature. The imbibition-related profile of the Atwhy1why3 mutant seeds, was characterised by decreased expression of hypoxia-related and oxygen metabolism genes even in the absence of aging. Seed aging further decreased the abundance of hypoxia-related and oxygen metabolism transcripts relative to the wild type. These findings suggest that the WHY1 and WHY3 proteins regulate the imbibition-induced responses to oxygen availability and hypoxia. Loss of WHY1 and WHY3 functions decreases the ability of Arabidopsis seeds to resist the adverse effects of seed aging.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Oxígeno/metabolismo , Longevidad/genética , Semillas/genética , Semillas/metabolismo , Hipoxia/metabolismo , Germinación/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
Successful germination and seedling establishment are important determinants of crop yields and plant survival in natural environments. Germination potential is compromised by suboptimal environmental conditions that result in seed ageing and high levels of genome damage. However, the mutagenic and growth inhibitory potential of DNA damage accumulated in seeds on subsequent seedling growth remains largely unknown. Arabidopsis seeds deficient in the chromosomal break repair factors DNA LIGASE 4 and DNA LIGASE 6 exhibited hypersensitivity to the effects of natural ageing, with reduced germination vigour and seedling biomass relative to wild type seed. Here, we identify that aged Arabidopsis seed display elevated levels of programmed cell death (PCD) in the root meristem which persists into seedling establishment, with higher levels of cell death in lines deficient in DNA double strand break repair. Reporter lines determined the effects of seed ageing on mutation levels and intrachromosomal recombination frequencies. Seed deterioration resulted in strikingly elevated levels of frameshift mutations and genome instability in germinated seedlings. Thus, elevated levels genome damage incurred in the seed stage of the plant life cycle potentially impacts significantly on subsequent plant development. Furthermore, the mutagenic effects of seed ageing has potentially long-term implications on the genome stability of plant populations and ecosystem fitness. Collectively, we identify genome damage accumulated in suboptimal quality seed impacts on subsequent plant growth and genome stability, with associated implications for crop yields and plant survival under changing climates.
Asunto(s)
Arabidopsis , Arabidopsis/metabolismo , Ecosistema , Semillas/metabolismo , Plantones/metabolismo , Germinación/genética , Inestabilidad Genómica , ADN Ligasas/genética , ADN Ligasas/metabolismoRESUMEN
Environmental stresses pose a significant threat to food security. Understanding the function of proteins that regulate plant responses to biotic and abiotic stresses is therefore pivotal in developing strategies for crop improvement. The WHIRLY (WHY) family of DNA-binding proteins are important in this regard because they fulfil a portfolio of important functions in organelles and nuclei. The WHY1 and WHY2 proteins function as transcription factors in the nucleus regulating phytohormone synthesis and associated growth and stress responses, as well as fulfilling crucial roles in DNA and RNA metabolism in plastids and mitochondria. WHY1, WHY2 (and WHY3 proteins in Arabidopsis) maintain organelle genome stability and serve as auxiliary factors for homologous recombination and double-strand break repair. Our understanding of WHY protein functions has greatly increased in recent years, as has our knowledge of the flexibility of their localization and overlap of functions but there is no review of the topic in the literature. Our aim in this review was therefore to provide a comprehensive overview of the topic, discussing WHY protein functions in nuclei and organelles and highlighting roles in plant development and stress responses. In particular, we consider areas of uncertainty such as the flexible localization of WHY proteins in terms of retrograde signalling connecting mitochondria, plastids, and the nucleus. Moreover, we identify WHY proteins as important targets in plant breeding programmes designed to increase stress tolerance and the sustainability of crop yield in a changing climate.
RESUMEN
The desiccated, quiescent state of seeds confers extended survival of the embryonic plant. However, accumulation of striking levels of genome damage in quiescence impairs germination and threatens plant survival. The mechanisms by which seeds mitigate this damage remain unclear. Here, we reveal that imbibed Arabidopsis seeds display high resistance to DNA damage, which is lost as seeds advance to germination, coincident with increasing cell cycle activity. In contrast to seedlings, we show that seeds minimize the impact of DNA damage by reducing meristem disruption and delaying SOG1-dependent programmed cell death. This promotes root growth early postgermination. In response to naturally accumulated DNA damage in aging seeds, SOG1 activates cell death postgermination. SOG1 activities are also important for promoting successful seedling establishment. These distinct cellular responses of seeds and seedlings are reflected by different DNA damage transcriptional profiles. Comparative analysis of DNA repair mutants identifies roles of the major genome maintenance pathways in germination but that the repair of cytotoxic chromosomal breaks is the most important for seed longevity. Collectively, these results indicate that high levels of DNA damage incurred in seeds are countered by low cell cycle activity, cell cycle checkpoints, and DNA repair, promoting successful seedling establishment. Our findings reveal insight into both the physiological significance of plant DNA damage responses and the mechanisms which maintain seed longevity, important for survival of plant populations in the natural environment and sustainable crop production under changing climates.
Asunto(s)
Arabidopsis , Daño del ADN , Germinación , Semillas , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Germinación/genética , Plantones/genética , Plantones/crecimiento & desarrollo , Semillas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Precise genome editing with CRISPR/Cas paves the way for many biochemical, biotechnological, and medical applications, and consequently, it may enable treatment of already known and still-to-be-found genetic diseases. Meanwhile, another rapidly emerging field-structural DNA nanotechnology-provides a customizable and modular platform for accurate positioning of nanoscopic materials, for e.g., biomedical uses. This addressability has just recently been applied in conjunction with the newly developed gene engineering tools to enable impactful, programmable nanotechnological applications. As of yet, self-assembled DNA nanostructures have been mainly employed to enhance and direct the delivery of CRISPR/Cas, but lately the groundwork has also been laid out for other intriguing and complex functions. These recent advances will be described in this perspective.
RESUMEN
DNA damage responses are crucial for plant growth under genotoxic stress. Accumulating evidence indicates that DNA damage responses differ between plant cell types. Here, quantitative shotgun phosphoproteomics provided high-throughput analysis of the DNA damage response network in callus cells. MS analysis revealed a wide network of highly dynamic changes in the phosphoprotein profile of genotoxin-treated cells, largely mediated by the ATAXIA TELANGIECTASIA MUTATED (ATM) protein kinase, representing candidate factors that modulate plant growth, development and DNA repair. A C-terminal dual serine target motif unique to H2AX in the plant lineage showed 171-fold phosphorylation that was absent in atm mutant lines. The physiological significance of post-translational DNA damage signalling to plant growth and survival was demonstrated using reverse genetics and complementation studies of h2ax mutants, establishing the functional role of ATM-mediated histone modification in plant growth under genotoxic stress. Our findings demonstrate the complexity and functional significance of post-translational DNA damage signalling responses in plants and establish the requirement of H2AX phosphorylation for plant survival under genotoxic stress.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Reparación del ADN , Histonas/metabolismo , Proteoma/metabolismo , Semillas/crecimiento & desarrollo , Estrés Fisiológico , Transportadoras de Casetes de Unión a ATP/genética , Envejecimiento/metabolismo , Envejecimiento/fisiología , Envejecimiento/efectos de la radiación , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Células Cultivadas , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Ontología de Genes , Germinación/genética , Germinación/efectos de la radiación , Histonas/química , Espectrometría de Masas , Fosforilación , Proteoma/genética , Proteoma/efectos de la radiación , Semillas/metabolismo , Semillas/fisiología , Semillas/efectos de la radiación , Serina/química , Serina/metabolismo , Transducción de Señal/efectos de la radiación , Estrés Fisiológico/genética , Estrés Fisiológico/efectos de la radiación , Rayos XRESUMEN
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.
RESUMEN
Carica papaya (papaya) seed germinate readily fresh from the fruit, but desiccation induces a dormant state. Dormancy can be released by exposure of the hydrated seed to a pulse of elevated temperature, typical of that encountered in its tropical habitat. Carica papaya is one of only a few species known to germinate in response to heat shock (HS) and we know little of the mechanisms that control germination in tropical ecosystems. Here we investigate the mechanisms that mediate HS-induced stimulation of germination in pre-dried and re-imbibed papaya seed. Exogenous gibberellic acid (GA3 ≥250 µM) overcame the requirement for HS to initiate germination. However, HS did not sensitise seeds to GA3, indicative that it may act independently of GA biosynthesis. Seed coat removal also overcame desiccation-imposed dormancy, indicative that resistance to radicle emergence is coat-imposed. Morphological and biomechanical studies identified that neither desiccation nor HS alter the physical structure or the mechanical strength of the seed coat. However, cycloheximide prevented both seed coat weakening and germination, implicating a requirement for de novo protein synthesis in both processes. The germination antagonist abscisic acid prevented radicle emergence but had no effect on papaya seed coat weakening. Desiccation therefore appears to reduce embryo growth potential, which is reversed by HS, without physically altering the mechanical properties of the seed coat. The ability to germinate in response to a HS may confer a competitive advantage to C. papaya, an opportunistic pioneer species, through detection of canopy removal in tropical forests.
Asunto(s)
Carica/metabolismo , Germinación/fisiología , Respuesta al Choque Térmico/fisiología , Semillas/metabolismo , Carica/fisiología , Cicloheximida/farmacología , Deshidratación , Germinación/efectos de los fármacos , Giberelinas/farmacología , Calor , Latencia en las Plantas/efectos de los fármacos , Latencia en las Plantas/fisiología , Reguladores del Crecimiento de las Plantas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Semillas/fisiologíaRESUMEN
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.
Asunto(s)
Arabidopsis/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Germinación/genética , Semillas/genética , Adaptación Fisiológica/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , ADN de Plantas/genética , Desecación , Inestabilidad Genómica , Mutación , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Transducción de Señal , Estrés Fisiológico , Factores de TiempoRESUMEN
Repair of DNA double-strand breaks (DSBs) by recombination pathways is essential for plant growth and fertility. The recombination endonuclease MRE11 plays important roles in sensing and repair of DNA DSBs. Here we demonstrate protein interaction between Arabidopsis MRE11 and the histone acetyltransferase TAF1, a TATA-binding protein Associated Factor (TAF) of the RNA polymerase II transcription initiation factor complex TFIID. Arabidopsis has two TAF1 homologues termed TAF1 and TAF1b and mutant taf1b lines are viable and fertile. In contrast, taf1 null mutations are lethal, demonstrating that TAF1 is an essential gene. Heterozygous taf1+/- plants display abnormal segregation of the mutant allele resulting from defects in pollen tube development, indicating that TAF1 is important for gamete viability. Characterization of an allelic series of taf1 lines revealed that hypomorphic mutants are viable but display developmental defects and reduced plant fertility. Hypersensitivity of taf1 mutants lacking the C-terminal bromodomain to X-rays and mitomycin C, but not to other forms of abiotic stress, established a specific role for TAF1 in plant DNA repair processes. Collectively these studies reveal a function for TAF1 in plant resistance to genotoxic stress, providing further insight into the molecular mechanisms of the DNA damage response in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Polen/fisiología , Factores de Transcripción/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica de las Plantas , Proteína Homóloga de MRE11 , Mitomicina/farmacología , Mutación , Tubo Polínico/genética , Tubo Polínico/metabolismo , Estructura Terciaria de Proteína , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Genoma de Planta , Germinación/genética , Fenómenos Fisiológicos de las Plantas/genética , Semillas/genética , Daño del ADN/genética , Reparación del ADN/genéticaRESUMEN
Non-homologous end joining (NHEJ) is the major model proposed for Agrobacterium T-DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA-PKcs, DNA ligase IV (LIG4), Ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR), play an important role in 'classical' (c)NHEJ. Other proteins, including histone H1 (HON1), XRCC1, and PARP1, participate in a 'backup' (b)NHEJ process. We examined transient and stable transformation frequencies of Arabidopsis thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, and the plant-specific DNA Ligase VI (LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild-type plants, suggesting enhanced T-DNA integration in these mutants. These results were confirmed using a promoter-trap transformation vector that requires T-DNA integration into the plant genome to activate a promoterless gusA (uidA) gene, by virus-induced gene silencing (VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T-DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, lig4, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T-DNA integration and transgene methylation. A double mutant (ku80/parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T-DNA integration. Thus, T-DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration.
Asunto(s)
Arabidopsis/genética , Reparación del ADN por Unión de Extremidades , Genoma de Planta/genética , Nicotiana/genética , Agrobacterium/genética , ADN Bacteriano/genética , ADN de Plantas/genética , Mutagénesis Insercional , Transformación GenéticaRESUMEN
DNA damage detection and repair take place in the context of chromatin, and histone proteins play important roles in these events. Post-translational modifications of histone proteins are involved in repair and DNA damage signalling processes in response to genotoxic stresses. In particular, acetylation of histones H3 and H4 plays an important role in the mammalian and yeast DNA damage response and survival under genotoxic stress. However, the role of post-translational modifications to histones during the plant DNA damage response is currently poorly understood. Several different acetylated H3 and H4 N-terminal peptides following X-ray treatment were identified using MS analysis of purified histones, revealing previously unseen patterns of histone acetylation in Arabidopsis. Immunoblot analysis revealed an increase in the relative abundance of the H3 acetylated N-terminus, and a global decrease in hyperacetylation of H4 in response to DNA damage induced by X-rays. Conversely, mutants in the key DNA damage signalling factor ATM (ATAXIA TELANGIECTASIA MUTATED) display increased histone acetylation upon irradiation, linking the DNA damage response with dynamic changes in histone modification in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Daño del ADN , Histonas/metabolismo , Acetilación/efectos de la radiación , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de la Ataxia Telangiectasia Mutada , ADN de Plantas/genética , ADN de Plantas/metabolismo , ADN de Plantas/efectos de la radiación , Histonas/química , Histonas/genética , Lisina/química , Datos de Secuencia Molecular , Mutación , Procesamiento Proteico-Postraduccional/efectos de la radiación , Espectrometría de Masas en TándemRESUMEN
DNA 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.
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Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Genoma de Planta/genética , Plantas/genética , Mutágenos/toxicidad , Desarrollo de la Planta , Plantas/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
DNA 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 ß-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.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , ADN Ligasas/genética , Semillas/genética , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Clonación Molecular , Frío , Daño del ADN , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Germinación/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Estrés Oxidativo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability. RESULTS: Knockout mutants of atlig1 are lethal. Therefore, RNAi lines with reduced levels of AtLIG1 were generated to allow the roles and importance of Arabidopsis DNA ligase 1 in DNA metabolism to be elucidated. Viable plants were fertile but displayed a severely stunted and stressed growth phenotype. Cell size was reduced in the silenced lines, whilst flow cytometry analysis revealed an increase of cells in S-phase in atlig1-RNAi lines relative to wild type plants. Comet assay analysis of isolated nuclei showed atlig1-RNAi lines displayed slower repair of single strand breaks (SSBs) and also double strand breaks (DSBs), implicating AtLIG1 in repair of both these lesions. CONCLUSION: Reduced levels of Arabidopsis DNA ligase 1 in the silenced lines are sufficient to support plant development but result in retarded growth and reduced cell size, which may reflect roles for AtLIG1 in both replication and repair. The finding that DNA ligase 1 plays an important role in DSB repair in addition to its known function in SSB repair, demonstrates the existence of a previously uncharacterised novel pathway, independent of the conserved NHEJ. These results indicate that DNA ligase 1 functions in both DNA replication and in repair of both ss and dsDNA strand breaks in higher plants.
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Arabidopsis/genética , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , ADN Ligasas/metabolismo , Reparación del ADN , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Tamaño de la Célula , Ensayo Cometa , ADN Ligasa (ATP) , Interferencia de ARNRESUMEN
Strong barriers prevent the financing of clinical information systems (CIS) in health care delivery system organizations in market segments serving disadvantaged patients. These segments include community health centers, public hospitals, unaffiliated rural hospitals, and some Medicaid-oriented solo and small-group medical practices. Policy interventions such as loans, grants, pay-for-performance and other reimbursement changes, and support services assistance will help lower these barriers. Without intervention, progress will be slow and worsen health care disparities between the advantaged and disadvantaged populations.
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Eficiencia Organizacional , Registros de Salud Personal , Evaluación de Procesos, Atención de Salud , HumanosRESUMEN
DNA damage in Arabidopsis thaliana seedlings results in upregulation of hundreds of genes. One of the earliest and highest levels of induction is displayed by a previously uncharacterized gene that we have termed X-ray induced 1 (XRI1). Analysis of plants carrying a null xri1 allele revealed two distinct requirements for this gene in plant fertility. XRI1 was important for the post-meiotic stages of pollen development, leading to inviability of xri(-) pollen and abnormal segregation of the mutant allele in heterozygous xri1(+/-) plants. In addition, XRI1 was essential for male and female meiosis, as indicated by the complete sterility of homozygous xri1 mutants due to extensive chromosome fragmentation visible in meiocytes. Abolition of programmed DNA double-strand breaks in a spo11-1 mutant background failed to rescue the DNA fragmentation of xri1 mutants, suggesting that XRI1 functions at an earlier stage than SPO11-1 does. Yeast two-hybrid studies identified an interaction between XRI1 and a novel component of the Arabidopsis MND1/AHP2 complex, indicating possible requirements for XRI1 in meiotic DNA repair.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Gametogénesis/genética , Meiosis/genética , Alelos , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efectos de la radiación , Clonación Molecular , Roturas del ADN de Doble Cadena , Fragmentación del ADN , Reparación del ADN , ADN de Plantas/genética , Flores/crecimiento & desarrollo , Meiosis/efectos de la radiación , Datos de Secuencia Molecular , Alineación de Secuencia , Rayos XRESUMEN
DNA double strand breaks (DSBs) are one of the most cytotoxic forms of DNA damage and must be repaired by recombination, predominantly via non-homologous joining of DNA ends (NHEJ) in higher eukaryotes. However, analysis of DSB repair kinetics of plant NHEJ mutants atlig4-4 and atku80 with the neutral comet assay shows that alternative DSB repair pathways are active. Surprisingly, these kinetic measurements show that DSB repair was faster in the NHEJ mutant lines than in wild-type Arabidopsis. Here we provide the first characterization of this KU-independent, rapid DSB repair pathway operating in Arabidopsis. The alternate pathway that rapidly removes the majority of DSBs present in nuclear DNA depends upon structural maintenance of chromosomes (SMC) complex proteins, namely MIM/AtRAD18 and AtRAD21.1. An absolute requirement for SMC proteins and kleisin for rapid repair of DSBs in Arabidopsis opens new insight into the mechanism of DSB removal in plants.
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Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/fisiología , Antibióticos Antineoplásicos/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Bleomicina/farmacología , Proteínas Cromosómicas no Histona/fisiología , Cromosomas de las Plantas/química , Cromosomas de las Plantas/metabolismo , Ensayo Cometa , Fragmentación del ADN/efectos de los fármacos , ADN de Plantas/efectos de los fármacos , ADN de Plantas/metabolismo , Relación Dosis-Respuesta a Droga , Recombinación Genética/fisiología , Factores de TiempoRESUMEN
In a 2004-05 study we found that five of six Community Health Centers (CHCs) used their Electronic Health Records (EHR) to improve clinical quality (technology enabled quality improvement (TEQI)); we also found that TEQI must be rapidly used for many conditions to justify the high EHR cost. We conducted a two year follow-up of CHCs to investigate changes in TEQI efforts. We found that while study CHCs maintained their TEQI activities, TEQI expansion was limited.