RESUMEN
Although the temporary presence of ribonucleotides in DNA is normal, their persistence represents a form of DNA damage. Here, we assess such damage and damage defense to DNA in plastids and mitochondria of maize. Shoot development proceeds from meristematic, non-pigmented cells containing proplastids and promitochondria at the leaf base to non-dividing green cells in the leaf blade containing mature organelles. The organellar DNAs (orgDNAs) become fragmented during this transition. Previously, orgDNA damage and damage defense of two types, oxidative and glycation, was described in maize, and now a third type, ribonucleotide damage, is reported. We hypothesized that ribonucleotide damage changes during leaf development and could contribute to the demise of orgDNAs. The levels of ribonucleotides and R-loops in orgDNAs and of RNase H proteins in organelles were measured throughout leaf development and in leaves grown in light and dark conditions. The data reveal that ribonucleotide damage to orgDNAs increased by about 2- to 5-fold during normal maize development from basal meristem to green leaf and when leaves were grown in normal light conditions compared to in the dark. During this developmental transition, the levels of the major agent of defense, RNase H, declined. The decline in organellar genome integrity during maize development may be attributed to oxidative, glycation, and ribonucleotide damages that are not repaired.
RESUMEN
Eukaryotic ribosomal DNA (rDNA) comprises tandem units of highly conserved coding genes separated by rapidly evolving spacer DNA. The spacers of all 12 species examined were filled with short direct repeats (DRs) and multiple long tandem repeats (TRs), completing the rDNA maps that previously contained unannotated and inadequately studied sequences. The external transcribed spacers also were filled with DRs and some contained TRs. We infer that the spacers arose from transposon insertion, followed by their imprecise excision, leaving short DRs characteristic of transposon visitation. The spacers provided a favored location for transposon insertion because they occupy loci containing hundreds to thousands of gene repeats. The spacers' primary cellular function may be to link one ribosomal RNA transcription unit to the next, whereas transposons flourish here because they have colonized the most frequently used part of the genome.
Asunto(s)
ARN Ribosómico , Secuencias Repetitivas de Ácidos Nucleicos , ADN Ribosómico/genética , ARN Ribosómico/genética , ADN IntergénicoRESUMEN
Oxidative damage to plant proteins, lipids, and DNA caused by reactive oxygen species (ROS) has long been studied. The damaging effects of reactive carbonyl groups (glycation damage) to plant proteins and lipids have also been extensively studied, but only recently has glycation damage to the DNA in plant mitochondria and plastids been reported. Here, we review data on organellar DNA maintenance after damage from ROS and glycation. Our focus is maize, where tissues representing the entire range of leaf development are readily obtained, from slow-growing cells in the basal meristem, containing immature organelles with pristine DNA, to fast-growing leaf cells, containing mature organelles with highly-fragmented DNA. The relative contributions to DNA damage from oxidation and glycation are not known. However, the changing patterns of damage and damage-defense during leaf development indicate tight coordination of responses to oxidation and glycation events. Future efforts should be directed at the mechanism by which this coordination is achieved.
RESUMEN
Shoot development in maize progresses from small, non-pigmented meristematic cells to expanded cells in the green leaf. During this transition, large plastid DNA (ptDNA) molecules in proplastids become fragmented in the photosynthetically-active chloroplasts. The genome sequences were determined for ptDNA obtained from Zea mays B73 plastids isolated from four tissues: base of the stalk (the meristem region); fully-developed first green leaf; first three leaves from light-grown seedlings; and first three leaves from dark-grown (etiolated) seedlings. These genome sequences were then compared to the Z. mays B73 plastid reference genome sequence that was previously obtained from green leaves. The assembled plastid genome was identical among these four tissues to the reference genome. Furthermore, there was no difference among these tissues in the sequence at and around the previously documented 27 RNA editing sites. There were, however, more sequence variants (insertions/deletions and single-nucleotide polymorphisms) for leaves grown in the dark than in the light. These variants were tightly clustered into two areas within the inverted repeat regions of the plastid genome. We propose a model for how these variant clusters could be generated by replication-transcription conflict.
RESUMEN
Shoot development in maize begins when meristematic, non-pigmented cells at leaf base stop dividing and proceeds toward the expanded green cells of the leaf blade. During this transition, promitochondria and proplastids develop into mature organelles and their DNA becomes fragmented. Changes in glycation damage during organelle development were measured for protein and DNA, as well as the glycating agent methyl glyoxal and the glycation-defense protein DJ-1 (known as Park7 in humans). Maize seedlings were grown under normal, non-stressful conditions. Nonetheless, we found that glycation damage, as well as defenses against glycation, follow the same developmental pattern we found previously for reactive oxygen species (ROS): as damage increases, damage-defense measures decrease. In addition, light-grown leaves had more glycation and less DJ-1 compared to dark-grown leaves. The demise of maize organellar DNA during development may therefore be attributed to both oxidative and glycation damage that is not repaired. The coordination between oxidative and glycation damage, as well as damage-response from the nucleus is also discussed.
Asunto(s)
ADN de Plantas/metabolismo , Orgánulos/metabolismo , Proteínas de Plantas/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Plantones/crecimiento & desarrollo , Zea mays/crecimiento & desarrollo , ADN de Plantas/genética , Orgánulos/genética , Proteínas de Plantas/genética , Proteína Desglicasa DJ-1/genética , Plantones/genética , Zea mays/genéticaRESUMEN
Maize shoot development progresses from non-pigmented meristematic cells at the base of the leaf to expanded and non-dividing green cells of the leaf blade. This transition is accompanied by the conversion of promitochondria and proplastids to their mature forms and massive fragmentation of both mitochondrial DNA (mtDNA) and plastid DNA (ptDNA), collectively termed organellar DNA (orgDNA). We measured developmental changes in reactive oxygen species (ROS), which at high concentrations can lead to oxidative stress and DNA damage, as well as antioxidant agents and oxidative damage in orgDNA. Our plants were grown under normal, non-stressful conditions. Nonetheless, we found more oxidative damage in orgDNA from leaf than stalk tissues and higher levels of hydrogen peroxide, superoxide, and superoxide dismutase in leaf than stalk tissues and in light-grown compared to dark-grown leaves. In both mitochondria and plastids, activities of the antioxidant enzyme peroxidase were higher in stalk than in leaves and in dark-grown than light-grown leaves. In protoplasts, the amount of the small-molecule antioxidants, glutathione and ascorbic acid, and catalase activity were also higher in the stalk than in leaf tissue. The data suggest that the degree of oxidative stress in the organelles is lower in stalk than leaf and lower in dark than light growth conditions. We speculate that the damaged/fragmented orgDNA in leaves (but not the basal meristem) results from ROS signaling to the nucleus to stop delivering DNA repair proteins to mature organelles producing large amounts of ROS.
RESUMEN
The structure of a chromosomal DNA molecule may influence the way in which it is replicated and inherited. For decades plastid DNA (ptDNA) was believed to be circular, with breakage invoked to explain linear forms found upon extraction from the cell. Recent evidence indicates that ptDNA in vivo consists of linear molecules with discrete termini, although these ends were not characterized. We report the sequences of two terminal regions, End1 and End2, for maize (Zea mays L.) ptDNA. We describe structural features of these terminal regions and similarities found in other plant ptDNAs. The terminal sequences are within inverted repeat regions (leading to four genomic isomers) and adjacent to origins of replication. Conceptually, stem-loop structures may be formed following melting of the double-stranded DNA ends. Exonuclease digestion indicates that the ends in maize are unobstructed, but tobacco (Nicotiana tabacum L.) ends may have a 5'-protein. If the terminal structure of ptDNA molecules influences the retention of ptDNA, the unprotected molecular ends in mature leaves of maize may be more susceptible to degradation in vivo than the protected ends in tobacco. The terminal sequences and cumulative GC skew profiles are nearly identical for maize, wheat (Triticum aestivum L.) and rice (Oryza sativa L.), with less similarity among other plants. The linear structure is now confirmed for maize ptDNA and inferred for other plants and suggests a virus-like recombination-dependent replication mechanism for ptDNA. Plastid transformation vectors containing the terminal sequences may increase the chances of success in generating transplastomic cereals.
Asunto(s)
Cromosomas de las Plantas , Replicación del ADN , ADN de Plantas/genética , Plastidios , Zea mays/genética , Secuencia de Bases , Homología de Secuencia de Ácido NucleicoRESUMEN
The DNA molecules in plastids and mitochondria of plants have been studied for over 40 years. Here, we review the data on the circular or linear form, replication, repair, and persistence of the organellar DNA (orgDNA) in plants. The bacterial origin of orgDNA appears to have profoundly influenced ideas about the properties of chromosomal DNA molecules in these organelles to the point of dismissing data inconsistent with ideas from the 1970s. When found at all, circular genome-sized molecules comprise a few percent of orgDNA. In cells active in orgDNA replication, most orgDNA is found as linear and branched-linear forms larger than the size of the genome, likely a consequence of a virus-like DNA replication mechanism. In contrast to the stable chromosomal DNA molecules in bacteria and the plant nucleus, the molecular integrity of orgDNA declines during leaf development at a rate that varies among plant species. This decline is attributed to degradation of damaged-but-not-repaired molecules, with a proposed repair cost-saving benefit most evident in grasses. All orgDNA maintenance activities are proposed to occur on the nucleoid tethered to organellar membranes by developmentally-regulated proteins.
RESUMEN
MAIN CONCLUSION: When compared to maize mesophyll cells, the plastid and mitochondrial DNAs in bundle sheath cells are less fragmented, less damaged, and contain fewer DNA polymerase-blocking impediments. Plants that conduct C4 photosynthesis differ from those that employ C3 photosynthesis with respect to leaf anatomy, biochemical pathways, and the proteins and RNA transcripts present in the leaf mesophyll (M) and bundle sheath (BS) cells. Here, we investigate the organellar DNA (orgDNA) from plastids and mitochondria in these two cell types. We use standard qPCR, long PCR, and DNA damage analysis to quantify the amount and quality of orgDNA in isolated M and BS cells of maize. When compared to M cells, BS cells have less orgDNA damage and a higher percentage of unimpeded orgDNA. In addition, the orgDNA is more fragmented in M than BS cells, although orgDNA in BS is subject to more in vitro repair. We suggest that the differences in molecular integrity of orgDNA in these two cells are due to higher levels of reactive oxygen species in M than BS cells.
Asunto(s)
ADN de Cloroplastos/genética , ADN Mitocondrial/genética , Zea mays/genética , Reparación del ADN , Genes de Plantas , Fotosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Zea mays/citología , Zea mays/fisiologíaRESUMEN
The amount and structural integrity of organellar DNAs change during plant development, although the mechanisms of change are poorly understood. Using PCR-based methods, we quantified DNA damage, molecular integrity, and genome copy number for plastid and mitochondrial DNAs of maize seedlings. A DNA repair assay was also used to assess DNA impediments. During development, DNA damage increased and molecules with impediments that prevented amplification by Taq DNA polymerase increased, with light causing the greatest change. DNA copy number values depended on the assay method, with standard real-time quantitative PCR (qPCR) values exceeding those determined by long-PCR by 100- to 1000-fold. As the organelles develop, their DNAs may be damaged in oxidative environments created by photo-oxidative reactions and photosynthetic/respiratory electron transfer. Some molecules may be repaired, while molecules with unrepaired damage may be degraded to non-functional fragments measured by standard qPCR but not by long-PCR.
Asunto(s)
Daño del ADN/genética , ADN Mitocondrial/genética , ADN de Plantas/genética , Dosificación de Gen , Plastidios/genética , Zea mays/crecimiento & desarrollo , Zea mays/genética , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Luz , Modelos Biológicos , Plastidios/efectos de la radiación , Reacción en Cadena de la Polimerasa , Plantones/genética , Plantones/efectos de la radiación , Zea mays/efectos de la radiaciónRESUMEN
For most eukaryotic organisms, the nuclear genomes of both parents are transmitted to the progeny following biparental inheritance. For mitochondria and chloroplasts, however, uniparental inheritance (UPI) is frequently observed. The maternal mode of inheritance for mitochondria in animals can be nearly absolute, suggesting an adaptive advantage for UPI. In other organisms, however, the mode of inheritance for mitochondria and chloroplasts can vary greatly even among strains of a species. Here, I review the data on the transmission of organellar DNA (orgDNA) from parent to progeny and the structure, copy number, and stability of orgDNA molecules. I propose that UPI is an incidental by-product of DNA abandonment, a process that lowers the metabolic cost of orgDNA repair.
Asunto(s)
Cloroplastos/genética , ADN de Cloroplastos/genética , ADN Mitocondrial/genética , Patrón de Herencia/genética , Mitocondrias/genética , Animales , Humanos , Mutación/genéticaRESUMEN
In maize and other grasses there is a developmental gradient from the meristematic cells at the base of the stalk to the differentiated cells at the leaf tip. This gradient presents an opportunity to investigate changes in mitochondrial DNA (mtDNA) that accompany growth under light and dark conditions, as done previously for plastid DNA. Maize mtDNA was analyzed by DAPI-DNA staining of individual mitochondria, gel electrophoresis/blot hybridization, and real-time qPCR. Both the amount and integrity of the mtDNA were found to decline with development. There was a 20-fold decline in mtDNA copy number per cell from the embryo to the light-grown leaf blade. The amount of DNA per mitochondrial particle was greater in dark-grown leaf blade (24 copies, on average) than in the light (2 copies), with some mitochondria lacking any detectable DNA. Three factors that influence the demise of mtDNA during development are considered: (1) the decision to either repair or degrade mtDNA molecules that are damaged by the reactive oxygen species produced as byproducts of respiration; (2) the generation of ATP by photophosphorylation in chloroplasts, reducing the need for respiratory-competent mitochondria; and (3) the shift in mitochondrial function from energy-generating respiration to photorespiration during the transition from non-green to green tissue.
Asunto(s)
ADN Mitocondrial/análisis , Mitocondrias/genética , Zea mays/crecimiento & desarrollo , Zea mays/genética , Aldehídos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Respiración de la Célula , Variaciones en el Número de Copia de ADN , ADN de Plantas/análisis , Oscuridad , Electroforesis en Gel de Campo Pulsado , Indoles , Luz , Microscopía Fluorescente , Mitocondrias/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/efectos de la radiación , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Coloración y Etiquetado , Factores de Tiempo , Zea mays/efectos de la radiaciónRESUMEN
Many areas of chloroplast research require methods that can assess the quality and quantity of chloroplast DNA (cpDNA). The study of chloroplast functions that depend on the proper maintenance and expression of the chloroplast genome, understanding cpDNA replication and repair, and the development of technologies for chloroplast transformation are just some of the disciplines that require the isolation of high-quality cpDNA. Arabidopsis thaliana offers several advantages for studying these processes because of the sizeable collection of mutants and natural varieties (accessions) available from stock centers and a broad community of researchers that has developed many other genetic resources. Several approaches for the isolation and quantification of cpDNA have been developed, but little consideration has been given to the strengths and weaknesses and the type of information obtained by each method, especially with respect to A. thaliana. Here, we provide protocols for obtaining high-quality cpDNA for PCR and other applications, and we evaluate several different isolation and analytical methods in order to build a robust framework for the study of cpDNA with this model organism.
Asunto(s)
Arabidopsis/metabolismo , Cloroplastos/genética , ADN de Cloroplastos/análisis , ADN de Cloroplastos/aislamiento & purificación , Biología Molecular/métodos , Fosfatasa Alcalina/metabolismo , Núcleo Celular/genética , Cetrimonio , Compuestos de Cetrimonio/metabolismo , Electroforesis en Gel de Campo Pulsado , Etidio/metabolismo , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Indoles/metabolismo , Microscopía Fluorescente , Hibridación de Ácido Nucleico , Reacción en Cadena en Tiempo Real de la Polimerasa , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coloración y EtiquetadoRESUMEN
DNA sequences similar to those in the organellar genomes are also found in the nucleus. These non-coding sequences may be co-amplified by PCR with the authentic organellar DNA sequences, leading to erroneous conclusions. To avoid this problem, we describe an experimental procedure to prevent amplification of this "promiscuous" DNA when total tissue DNA is used with PCR. First, primers are designed for organelle-specific sequences using a bioinformatics method. These primers are then tested using methylation-sensitive PCR. The method is demonstrated for both end-point and real-time PCR with Zea mays, where most of the DNA sequences in the organellar genomes are also present in the nucleus. We use this procedure to quantify those nuclear DNA sequences that are near-perfect replicas of organellar DNA. This method should be useful for applications including phylogenetic analysis, organellar DNA quantification and clinical testing.
Asunto(s)
Núcleo Celular/química , ADN Mitocondrial/análisis , Plastidios/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Núcleo Celular/genética , ADN Mitocondrial/genética , Zea mays/química , Zea mays/genéticaRESUMEN
In maize (Zea mays L.), chloroplast development progresses from the basal meristem to the mature leaf tip, and light is required for maturation to photosynthetic competence. During chloroplast greening, it was found that chloroplast DNA (cpDNA) is extensively degraded, falling to undetectable levels in many individual chloroplasts for three maize cultivars, as well as Zea mexicana (the ancestor of cultivated maize) and the perennial species Zea diploperennis. In dark-grown maize seedlings, the proplastid-to-etioplast transition is characterized by plastid enlargement, cpDNA replication, and the retention of high levels of cpDNA. When dark-grown seedlings are transferred to white light, the DNA content per plastid increases slightly during the first 4 h of illumination and then declines rapidly to a minimum at 24 h during the etioplast-to-chloroplast transition. Plastid autofluorescence (from chlorophyll) continues to increase as cpDNA declines, whereas plastid size remains constant. It is concluded that the increase in cpDNA that accompanies plastid enlargement is a consequence of cell and leaf growth, rather than illumination, whereas light stimulates photosynthetic capacity and cpDNA instability. When cpDNA from total tissue was monitored by blot hybridization and real-time quantitative PCR, no decline following transfer from dark to light was observed. The lack of agreement between DNA per plastid and cpDNA per cell may be attributed to nupts (nuclear sequences of plastid origin).
Asunto(s)
ADN de Cloroplastos/metabolismo , Luz , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/efectos de la radiación , Plastidios/genética , Zea mays/crecimiento & desarrollo , Zea mays/efectos de la radiación , Secuencia de Bases , Núcleo Celular/genética , Núcleo Celular/efectos de la radiación , Clorofila/metabolismo , ADN de Cloroplastos/genética , Fluorescencia , Dosificación de Gen/efectos de la radiación , Genoma de Planta/genética , Hojas de la Planta/genética , Plastidios/efectos de la radiación , Mapeo Restrictivo , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Plantones/efectos de la radiación , Especificidad de la Especie , Zea mays/genéticaRESUMEN
In this issue of Molecular Cell, Gerhold et al. (2010) find no circular DNA during mitochondrial DNA (mtDNA) replication in the aerobic yeast Candida albicans, a result with important implications for mtDNA replication in Saccharomyces cerevisiae.
RESUMEN
BACKGROUND: Several proposals have been made to explain the rise of multicellular life forms. An internal environment can be created and controlled, germ cells can be protected in novel structures, and increased organismal size allows a "division of labor" among cell types. These proposals describe advantages of multicellular versus unicellular organisms at levels of organization at or above the individual cell. I focus on a subsequent phase of evolution, when multicellular organisms initiated the process of development that later became the more complex embryonic development found in animals and plants. The advantage here is realized at the level of the mitochondrion and chloroplast. HYPOTHESIS: The extreme instability of DNA in mitochondria and chloroplasts has not been widely appreciated even though it was first reported four decades ago. Here, I show that the evolutionary success of multicellular animals and plants can be traced to the protection of organellar DNA. Three stages are envisioned. Sequestration allowed mitochondria and chloroplasts to be placed in "quiet" germ line cells so that their DNA is not exposed to the oxidative stress produced by these organelles in "active" somatic cells. This advantage then provided Opportunity, a period of time during which novel processes arose for signaling within and between cells and (in animals) for cell-cell recognition molecules to evolve. Development then led to the enormous diversity of animals and plants. IMPLICATIONS: The potency of a somatic stem cell is its potential to generate cell types other than itself, and this is a systems property. One of the biochemical properties required for stemness to emerge from a population of cells might be the metabolic quiescence that protects organellar DNA from oxidative stress.
Asunto(s)
Evolución Biológica , ADN de Cloroplastos/genética , ADN Mitocondrial/genética , Eucariontes/genética , Animales , Modelos Teóricos , Plantas/genéticaRESUMEN
Although our understanding of mechanisms of DNA repair in bacteria and eukaryotic nuclei continues to improve, almost nothing is known about the DNA repair process in plant organelles, especially chloroplasts. Since the RecA protein functions in DNA repair for bacteria, an analogous function may exist for chloroplasts. The effects on chloroplast DNA (cpDNA) structure of two nuclear-encoded, chloroplast-targeted homologues of RecA in Arabidopsis were examined. A homozygous T-DNA insertion mutation in one of these genes (cpRecA) resulted in altered structural forms of cpDNA molecules and a reduced amount of cpDNA, while a similar mutation in the other gene (DRT100) had no effect. Double mutants exhibited a similar phenotype to cprecA single mutants. The cprecA mutants also exhibited an increased amount of single-stranded cpDNA, consistent with impaired RecA function. After four generations, the cprecA mutant plants showed signs of reduced chloroplast function: variegation and necrosis. Double-stranded breaks in cpDNA of wild-type plants caused by ciprofloxacin (an inhibitor of Escherichia coli gyrase, a type II topoisomerase) led to an alteration of cpDNA structure that was similar to that seen in cprecA mutants. It is concluded that the process by which damaged DNA is repaired in bacteria has been retained in their endosymbiotic descendent, the chloroplast.
Asunto(s)
Arabidopsis/metabolismo , Cloroplastos/genética , ADN de Cloroplastos/metabolismo , Rec A Recombinasas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Cloroplastos/metabolismo , ADN Bacteriano/genética , ADN de Cloroplastos/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genoma de Planta/genética , Mutación/genética , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In this review, the controversy regarding the preservation or degradation of chloroplast DNA (cpDNA) as chloroplasts develop their photosynthetic capacity and leaves reach maturity is addressed. A constant amount of cpDNA during maturity might be expected in order to support photosynthesis over the lifespan of the leaf. Nevertheless, a decline in cpDNA during leaf development was found for all seven plant species investigated. Initial measurements showed that Arabidopsis was similar to the other seven. The controversy arose with two recent studies concluding that the amount of cpDNA remains constant as Arabidopsis leaves mature. These authors proposed that the observation of Arabidopsis chloroplasts with undetectable levels of DNA was an artefact, although the most recent data support the original findings. If the amount of cpDNA remains constant, then Arabidopsis is atypical and would not serve as a good model for chloroplast development. It is shown that the apparently contradictory data may be attributed to methodology and the choice of leaves to be compared. Thus, it is concluded that the controversy can be resolved, Arabidopsis can serve as a representative model, and cpDNA degradation is a common event in chloroplast development.