RESUMO
An ultimate goal of evolutionary biology is the prediction and experimental verification of adaptive trajectories on macroevolutionary timescales. This aim has rarely been achieved for complex biological systems, as models usually lack clear correlates of organismal fitness. Here, we simulate the fitness landscape connecting two carbon fixation systems: C3 photosynthesis, used by most plant species, and the C4 system, which is more efficient at ambient CO2 levels and elevated temperatures and which repeatedly evolved from C3. Despite extensive sign epistasis, C4 photosynthesis is evolutionarily accessible through individually adaptive steps from any intermediate state. Simulations show that biochemical subtraits evolve in modules; the order and constitution of modules confirm and extend previous hypotheses based on species comparisons. Plant-species-designated C3-C4 intermediates lie on predicted evolutionary trajectories, indicating that they indeed represent transitory states. Contrary to expectations, we find no slowdown of adaptation and no diminishing fitness gains along evolutionary trajectories.
Assuntos
Evolução Biológica , Fotossíntese , Plantas/genética , Adaptação Fisiológica , Ciclo do Carbono , Epistasia Genética , Evolução Molecular , Aptidão Genética , Mutação , Fenômenos Fisiológicos Vegetais , Plantas/classificaçãoRESUMO
A 3 yr old female spayed Labrador retriever was referred for the treatment of a chronic oropharyngeal stick injury. After computed tomography scan evaluation, the cervical area was explored surgically and a right-sided cervical abscess that contained a wooden stick was identified adjacent to the vagosympathetic trunk and carotid artery. The ipsilateral mandibular salivary gland was resected concurrently given its abnormal appearance, and histology confirmed inflammation and necrosis of the gland, which was suspected to be due to direct trauma from the foreign body. The clinical signs initially improved but then recurred, and a follow-up computed tomography scan was suggestive of sialadenosis or sialadenitis in the right parotid, zygomatic, and molar salivary glands. A presumptive diagnosis of sialadenosis was made and a course of phenobarbital was initiated. The clinical signs resolved completely within a few days, and there was no recurrence several months after termination of the phenobarbital treatment. This is the first case report of presumptive sialadenosis in a dog as a suspected complication of an oropharyngeal stick injury. Informed consent was obtained from the owner of the dog and the patient was managed according to contemporary standards of care.
Assuntos
Doenças do Cão , Sialadenite , Cães , Feminino , Animais , Doenças do Cão/tratamento farmacológico , Sialadenite/diagnóstico , Sialadenite/veterinária , Sialadenite/patologia , Orofaringe/lesões , Orofaringe/patologia , Fenobarbital , Glândula Parótida/patologiaRESUMO
BACKGROUND: Cellular events during meiosis can differ between inbred lines in maize. Substantial differences in the average numbers of chiasmata and double-strand breaks (DSBs) per meiotic cell have been documented among diverse inbred lines of maize: CML228, a tropical maize inbred line, B73 and Mo17, temperate maize lines. To determine if gene expression might explain these observed differences, an RNA-Seq experiment was performed on CML228 male meiocytes which was compared to B73 and Mo17 male meiocytes, where plants were grown in the same controlled environment. RESULTS: We found that a few DSB-repair/meiotic genes which promote class I crossovers (COs) and the Zyp1 gene which limits newly formed class I COs were up-regulated, whereas Mus81 homolog 2 which promotes class II COs was down-regulated in CML228. Although we did not find enriched gene ontology (GO) categories directly related to meiosis, we found that GO categories in membrane, localization, proteolysis, energy processes were up-regulated in CML228, while chromatin remodeling, epigenetic regulation, and cell cycle related processes including meiosis related cell cycle processes were down-regulated in CML228. The degree of similarity in expression patterns between the three maize lines reflect their genetic relatedness: B73 and Mo17 had similar meiotic expressions and CML228 had a more distinct expression profile. CONCLUSIONS: We found that meiotic related genes were mostly conserved among the three maize inbreds except for a few DSB-repair/meiotic genes. The findings that the molecular players in limiting class I CO formation (once CO assurance is achieved) were up-regulated and those involved in promoting class II CO formation were down-regulated in CML228 agree with the lower chiasmata number observed in CML228 previously. In addition, epigenetics such as chromatin remodeling and histone modification might play a role. Transport and energy-related processes was up-regulated and Cyclin13 was down-regulated in CML228. The direction of gene expression of these processes agree with that previously found in meiotic tissues compared with vegetative tissues. In summary, we used different natural maize inbred lines from different climatic conditions and have shown their differences in expression landscape in male meiocytes.
Assuntos
Quebras de DNA de Cadeia Dupla , Zea mays , Epigênese Genética , Meiose/genética , Recombinação Genética , Transcriptoma , Zea mays/metabolismoRESUMO
C4 photosynthetic plants have evolved from C3 ancestors and are characterized by differential expression of several hundred genes. Strict compartmentalization of key C4 enzymes either to mesophyll (M) or bundle sheath cells is considered a crucial step towards the evolution of C4 photosynthesis. In this study, we demonstrate that the 5'-flanking sequences of the C4 type phosphoenolpyruvate carboxylase (Ppc) gene from three C4 grass species could drive M-cell-specific expression of a reporter gene in rice. In addition to that, we identified about 450 bp (upstream of their transcription start site) of the analyzed C4 Ppc promoters contain all the essential regulatory elements for driving M-cell-specific expression in rice leaves. Importantly, four motifs of conserved nucleotide sequences (CNSs) were also determined, which are essential for the activity of the promoter. A putative interaction between the CNSs and an unknown upstream element(s) is required for driving M-cell-specific expression. This work identifies the evolutionary conservation of C4 Ppc regulatory mechanisms of multiple closely related C4 grass species.
Assuntos
Células do Mesofilo/metabolismo , Regiões Promotoras Genéticas/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese/genética , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
In an effort to identify genetic regulators for the cell ontogeny around the veins in Arabidopsis thaliana leaves, an activation-tagged mutant line with altered leaf morphology and altered bundle sheath anatomy was characterized. This mutant had a small rosette area with wrinkled leaves and chlorotic leaf edges, as well as enhanced chloroplast numbers in the (pre-)bundle sheath tissue. It had a bundle-specific promoter from the gene GLYCINE DECARBOXYLASE SUBUNIT-T from the C4 species Flaveria trinervia (GLDTFt promoter) inserted in the coding region of the transcriptional repressor NAC052, functioning in H3K4 demethylation, in front of an alternative start codon in-frame with the natural start codon. Reconstruction of the mutation event of our activation-tagged line by creating a line expressing an N-terminally truncated sequence of NAC052 under control of the GLDTFt promoter confirmed the involvement of NAC052 in leaf development. Our study not only reveals leaf anatomic and transcriptomic effects of an N-terminally truncated NAC052 under control of the GLDTFt promoter, but also identifies NAC052 as a novel genetic regulator of leaf development.
Assuntos
Arabidopsis , Flaveria , Arabidopsis/genética , Desmetilação , Fotossíntese , Folhas de Planta/genéticaRESUMO
Persistent and mobile organic substances (PM substances) are a threat to the quality of our water resources. While screening studies revealed widespread occurrence of many PM substances, rapid trace analytical methods for their quantification in large sample sets are missing. We developed a quick and generic analytical method for highly mobile analytes in surface water, groundwater, and drinking water samples based on enrichment through azeotrope evaporation (4 mL water and 21 mL acetonitrile), supercritical fluid chromatography (SFC) coupled to high-resolution mass spectrometry (HRMS), and quantification using a compound-specific correction factor for apparent recovery. The method was validated using 17 PM substances. Sample preparation recoveries were between 60 and 110% for the vast majority of PM substances. Strong matrix effects (most commonly suppressive) were observed, necessitating a correction for apparent recoveries in quantification. Apparent recoveries were neither concentration dependent nor dependent on the water matrix (surface or drinking water). Method detection and quantification limits were in the single- to double-digit ng L-1 ranges, precision expressed as relative standard deviation of quadruplicate quantifications was on average < 10%, and trueness experiments showed quantitative results within ± 30% of the theoretical value in 77% of quantifications. Application of the method to surface water, groundwater, raw water, and finished drinking water revealed the presence of acesulfame and trifluoromethanesulfonic acid up to 70 and 19 µg L-1, respectively. Melamine, diphenylguanidine, p-dimethylbenzenesulfonic acid, and 4-hydroxy-1-(2-hydroxyethyl)-2,2,6,6-tetramethylpiperidine were found in high ng L-1 concentrations. Graphical abstract.
RESUMO
Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA de Plantas/genética , Genoma de Planta , Meiose , Zea mays/genética , Mapeamento Cromossômico , DNA de Plantas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Reparo de DNA por Recombinação , Sequências de Repetição em Tandem , Zea mays/metabolismoRESUMO
Following publication of the original article [1], a reader spotted an incorrect citation of the reference 14 [2] in the 'Background'. The male meiocyte isolation work described in this article [2] was carried out in rice and not in Brassica as originally stated in the 'Background' [1]. Thus, the following amendment to the Background section should be noted.
RESUMO
BACKGROUND: Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. RESULTS: We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. CONCLUSION: The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.
Assuntos
Separação Celular/métodos , Prófase Meiótica I , Células Vegetais , Triticum/citologia , Flores/citologia , Flores/ultraestrutura , Células Vegetais/ultraestruturaRESUMO
CORRECTION: Following publication of the original article [1], the authors reported that the number of genes overlaying the bar graph in Fig. 3A were incorrectly counted and inserted (i.e. including a title tile, and in inverse order). The corrected numbers are below and match with the listed genes supplied in Additional File: Table S2.
RESUMO
DNA methylation and histone modifications are epigenetic changes on a DNA molecule that alter the three-dimensional (3D) structure locally as well as globally, impacting chromatin looping and packaging on a larger scale. Epigenetic marks thus inform higher-order chromosome organization and placement in the nucleus. Conventional epigenetic marks are joined by chromatin modifiers like cohesins, condensins and membrane-anchoring complexes to support particularly 3D chromosome organization. The most popular consequences of epigenetic modifications are gene expression changes, but chromatin modifications have implications beyond this, particularly in actively dividing cells and during sexual reproduction. In this opinion paper, we will focus on epigenetic mechanisms and chromatin modifications during meiosis as part of plant sexual reproduction where 3D management of chromosomes and re-organization of chromatin are defining features and prime tasks in reproductive cells, not limited to modulating gene expression. Meiotic chromosome organization, pairing and synapsis of homologous chromosomes as well as distribution of meiotic double-strand breaks and resulting crossovers are presumably highly influenced by epigenetic mechanisms. Special mobile small RNAs have been described in anthers, where these so-called phasiRNAs seem to direct DNA methylation in meiotic cells. Intriguingly, many of the mentioned developmental processes make use of epigenetic changes and small RNAs in a manner other than gene expression changes. Widening our approaches and opening our mind to thinking three-dimensionally regarding epigenetics in plant development holds high promise for new discoveries and could give us a boost for further knowledge.
Assuntos
Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Plantas/genética , Cromatina/química , Cromatina/genética , Cromossomos de Plantas/ultraestrutura , Epigênese Genética , Histonas , Meiose , Células Vegetais/fisiologia , Desenvolvimento Vegetal/genética , Polinização , Processamento de Proteína Pós-Traducional , RNA de Plantas/genéticaRESUMO
The first two reactions of C4 photosynthesis are catalysed by carbonic anhydrase (CA) and phosphoenolpyruvate carboxylase (PEPC) in the leaf mesophyll (M) cell cytosol. Translatome experiments using a tagged ribosomal protein expressed under the control of M and bundle-sheath (BS) cell-specific promoters showed transcripts encoding CA3 from the C4 species Flaveria bidentis were highly enriched in polysomes from M cells relative to those of the BS. Localisation experiments employing a CA3-green fluorescent protein fusion protein showed F. bidentis CA3 is a cytosolic enzyme. A motif showing high sequence homology to that of the Flaveria M expression module 1 (MEM1) element was identified approximately 2 kb upstream of the F. bidentis and F. trinervia ca3 translation start sites. MEM1 is located in the promoter of C4 Flaveria ppcA genes, which encode the C4-associated PEPC, and is necessary for M-specific expression. No MEM1-like sequence was found in the 4 kb upstream of the C3 species F. pringlei ca3 translation start site. Promoter-reporter fusion experiments demonstrated the region containing the ca3 MEM1-like element also directs M-specific expression. These results support the idea that a common regulatory switch drives the expression of the C4 Flaveria ca3 and ppcA1 genes specifically in M cells.
Assuntos
Flaveria/enzimologia , Regulação da Expressão Gênica de Plantas , Células do Mesofilo/enzimologia , Sequência de Bases , Flaveria/genética , Dados de Sequência MolecularRESUMO
Meiosis marks the transition from the sporophyte to the gametophyte generation in the life cycle of flowering plants, and creates genetic variations through homologous recombination. In most flowering plants, meiosis is highly synchronized within each anther, which is significant for efficient fertilization. To date, little is known about the molecular mechanisms of entry into meiosis and exit from it, and only a few genes in Arabidopsis have been characterized with a role in regulating meiotic progression. In this study, we report the functional characterization of a plant-specific basic helix-loop-helix (bHLH) protein, FEHLSTART (FST), a defect in which leads to premature meiotic entry and asynchronous meiosis, and results in decreased seed yield. Investigation of the time course of meiosis showed that the onset of leptotene, the first stage of prophase I, frequently occurred earlier in fst-1 than in the wild type. Asynchronous meiosis followed, which could manifest in the disruption of regular spindle structures and symmetric cell divisions in fst-1 mutants during the meiosis I/II transition. In accordance with frequently accelerated meiotic entry, whole-transcriptome analysis of fst-1 anthers undergoing meiosis revealed that 19 circadian rhythm genes were affected and 47 pollen-related genes were prematurely expressed at a higher level. Taken together, we propose that FST is required for normal meiotic entry and the establishment of meiotic synchrony.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Perfilação da Expressão Gênica/métodos , Meiose/genética , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/classificação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Filogenia , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
C4 photosynthesis is nature's most efficient answer to the dual activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the resulting loss of CO(2) by photorespiration. Gly decarboxylase (GDC) is the key component of photorespiratory CO(2) release in plants and is active in all photosynthetic tissues of C(3) plants, but only in the bundle sheath cells of C(4) plants. The restriction of GDC to the bundle sheath is assumed to be an essential and early step in the evolution of C(4) photosynthesis, leading to a photorespiratory CO(2) concentrating mechanism. In this study, we analyzed how the P-protein of GDC (GLDP) became restricted to the bundle sheath during the transition from C(3) to C(4) photosynthesis in the genus Flaveria. We found that C(3) Flaveria species already contain a bundle sheath-expressed GLDP gene in addition to a ubiquitously expressed second gene, which became a pseudogene in C(4) Flaveria species. Analyses of C(3)-C(4) intermediate Flaveria species revealed that the photorespiratory CO(2) pump was not established in one single step, but gradually. The knowledge gained by this study sheds light on the early steps in C(4) evolution.
Assuntos
Flaveria/metabolismo , Glicina Desidrogenase (Descarboxilante)/metabolismo , Fotossíntese , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Dióxido de Carbono/metabolismo , Evolução Molecular , Flaveria/classificação , Flaveria/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glicina Desidrogenase (Descarboxilante)/classificação , Glicina Desidrogenase (Descarboxilante)/genética , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Consumo de Oxigênio/efeitos da radiação , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Splicing de RNA , Ribulose-Bifosfato Carboxilase/genética , Especificidade da EspécieRESUMO
The mitochondrial Gly decarboxylase complex (GDC) is a key component of the photorespiratory pathway that occurs in all photosynthetically active tissues of C(3) plants but is restricted to bundle sheath cells in C(4) species. GDC is also required for general cellular C(1) metabolism. In the Asteracean C(4) species Flaveria trinervia, a single functional GLDP gene, GLDPA, encodes the P-subunit of GDC, a decarboxylating Gly dehydrogenase. GLDPA promoter reporter gene fusion studies revealed that this promoter is active in bundle sheath cells and the vasculature of transgenic Flaveria bidentis (C(4)) and the Brassicacean C(3) species Arabidopsis thaliana, suggesting the existence of an evolutionarily conserved gene regulatory system in the bundle sheath. Here, we demonstrate that GLDPA gene regulation is achieved by an intricate interplay of transcriptional and posttranscriptional mechanisms. The GLDPA promoter is composed of two tandem promoters, P(R2) and P(R7), that together ensure a strong bundle sheath expression. While the proximal promoter (P(R7)) is active in the bundle sheath and vasculature, the distal promoter (P(R2)) drives uniform expression in all leaf chlorenchyma cells and the vasculature. An intron in the 5' untranslated leader of P(R2)-derived transcripts is inefficiently spliced and apparently suppresses the output of P(R2) by eliciting RNA decay.
Assuntos
Flaveria/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Flaveria/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Dados de Sequência Molecular , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genéticaRESUMO
BACKGROUND: A major step in the higher plant life cycle is the decision to leave the mitotic cell cycle and begin the progression through the meiotic cell cycle that leads to the formation of gametes. The molecular mechanisms that regulate this transition and early meiosis remain largely unknown. To gain insight into gene expression features during the initiation of meiotic recombination, we profiled early prophase I meiocytes from maize (Zea mays) using capillary collection to isolate meiocytes, followed by RNA-seq. RESULTS: We detected ~2,000 genes as preferentially expressed during early meiotic prophase, most of them uncharacterized. Functional analysis uncovered the importance of several cellular processes in early meiosis. Processes significantly enriched in isolated meiocytes included proteolysis, protein targeting, chromatin modification and the regulation of redox homeostasis. The most significantly up-regulated processes in meiocytes were processes involved in carbohydrate metabolism. Consistent with this, many mitochondrial genes were up-regulated in meiocytes, including nuclear- and mitochondrial-encoded genes. The data were validated with real-time PCR and in situ hybridization and also used to generate a candidate maize homologue list of known meiotic genes from Arabidopsis. CONCLUSIONS: Taken together, we present a high-resolution analysis of the transcriptome landscape in early meiosis of an important crop plant, providing support for choosing genes for detailed characterization of recombination initiation and regulation of early meiosis. Our data also reveal an important connection between meiotic processes and altered/increased energy production.
Assuntos
Meiose/genética , Transcriptoma/genética , Zea mays/citologia , Zea mays/genética , Simulação por Computador , Elementos de DNA Transponíveis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Genes Mitocondriais , Estudos de Associação Genética , Hibridização In Situ , Endogamia , Mitocôndrias/genética , Pólen/citologia , Pólen/metabolismo , Edição de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Plântula/genética , Análise de Sequência de RNA , Regulação para Cima/genéticaRESUMO
In recent years, multiple factors involved in DNA double-strand break (DSB) repair have been characterised in Arabidopsis thaliana. Using homologous sequences in somatic cells, DSBs are mainly repaired by two different pathways: synthesis-dependent strand annealing (SDSA) and single-strand annealing (SSA). By applying recombination substrates in which recombination is initiated by the induction of a site-specific DSB by the homing endonuclease I-SceI, we were able to characterise the involvement of different factors in both pathways. The nucleases MRE11 and COM1, both involved in DSB end processing, were not required for either SDSA or SSA in our assay system. Both SDSA and SSA were even more efficient without MRE11, in accordance with the fact that a loss of MRE11 might negatively affect the efficiency of non-homologous end joining. Loss of the classical recombinase RAD51 or its two paralogues RAD51C and XRCC3, as well as the SWI2/SNF2 remodelling factor RAD54, resulted in a drastic deficiency in SDSA but had hardly any influence on SSA, confirming that a strand exchange reaction is only required for SDSA. The helicase FANCM, which is postulated to be involved in the stabilisation of recombination intermediates, is surprisingly not only needed for SDSA but to a lesser extent also for SSA. Both SSA and SDSA were affected only weakly when the SMC6B protein, implicated in sister chromatid recombination, was absent, indicating that SSA and SDSA are in most cases intrachromatid recombination reactions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Proteína Homóloga a MRE11 , Plantas Geneticamente Modificadas , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Recombinação GenéticaRESUMO
Complex DNA structures, such as double Holliday junctions and stalled replication forks, arise during DNA replication and DNA repair. Factors processing these intermediates include the endonuclease MUS81, helicases of the RecQ family, and the yeast SNF2 ATPase RAD5 and its Arabidopsis thaliana homolog RAD5A. By testing sensitivity of mutant plants to DNA-damaging agents, we defined the roles of these factors in Arabidopsis. rad5A recq4A and rad5A mus81 double mutants are more sensitive to cross-linking and methylating agents, showing that RAD5A is required for damage-induced DNA repair, independent of MUS81 and RECQ4A. The lethality of the recq4A mus81 double mutant indicates that MUS81 and RECQ4A also define parallel DNA repair pathways. The recq4A/mus81 lethality is suppressed by blocking homologous recombination (HR) through disruption of RAD51C, showing that RECQ4A and MUS81 are required for processing recombination-induced aberrant intermediates during replication. Thus, plants possess at least three different pathways to process DNA repair intermediates. We also examined HR-mediated double-strand break (DSB) repair using recombination substrates with inducible site-specific DSBs: MUS81 and RECQ4A are required for efficient synthesis-dependent strand annealing (SDSA) but only to a small extent for single-strand annealing (SSA). Interestingly, RAD5A plays a significant role in SDSA but not in SSA.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , DNA Helicases/metabolismo , Reparo do DNA , Endonucleases/metabolismo , Recombinação Genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA de Plantas/genética , Endonucleases/genética , Dados de Sequência Molecular , MutaçãoRESUMO
BRCA1 is a well-known tumor suppressor protein in mammals, involved in multiple cellular processes such as DNA repair, chromosome segregation and chromatin remodeling. Interestingly, homologs of BRCA1 and several of its complex partners are also found in plants. As the respective mutants are viable, in contrast to mammalian mutants, detailed analyses of their biological role is possible. Here we demonstrate that the model plant Arabidopsis thaliana harbors two homologs of the mammalian BRCA1 interaction partner BRCC36, AtBRCC36A and AtBRCC36B. Mutants of both genes as well as the double mutants are fully fertile and show no defects in development. We were able to show that mutation of one of the homologs, AtBRCC36A, leads to a severe defect in intra- and interchromosomal homologous recombination (HR). A HR defect is also apparent in Atbrca1 mutants. As the Atbrcc36a/Atbrca1 double mutant behaves like the single mutants of AtBRCA1 and AtBRCC36A both proteins seem to be involved in a common pathway in the regulation of HR. AtBRCC36 is also epistatic to AtBRCA1 in DNA crosslink repair. Upon genotoxic stress, AtBRCC36A is transferred into the nucleus.
Assuntos
Proteínas de Arabidopsis/fisiologia , Proteína BRCA1/fisiologia , Reparo do DNA , Epistasia Genética , Recombinação Genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteína BRCA1/genética , Bleomicina/toxicidade , Dados de Sequência Molecular , Proteínas Nucleares/análise , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Homologous recombination, together with selection, laid the foundation for traditional plant breeding. The recombination process that takes place during meiotic cell division is crucial for the creation of novel variations of highly desired traits by breeders. Gaining control over this process is important for molecular breeding to achieve more precise, large-scale and quicker plant improvement. As conventional ubiquitous promoters are neither tissue-specific nor efficient in driving gene expression in meiocytes, promoters with high meiotic activities are potential candidates for manipulating the recombination process. So far, only a few meiotically-active promoters have been reported. Recently developed techniques to profile the transcriptome landscape of isolated meiocytes provided the means to discover promoters from genes that are actively expressed in meiosis. RESULTS: In a screen for meiotically-active promoters, we examined ten promoter sequences that are associated with novel meiotic candidate genes. Each promoter was tested by expressing a GFP reporter gene in Arabidopsis. Characterization of regulatory regions revealed that these meiotically-active promoters possessed conserved motifs and motif arrangement. Some of the promoters unite optimal properties which are invaluable for meiosis-directed studies such as delivering specific gene expression in early meiosis I and/or meiosis II. Furthermore, the examination of homologs of the corresponding genes within green plants points to a great potential of applying the information from Arabidopsis to other species, especially crop plants. CONCLUSIONS: We identified ten novel meiotically-active promoters; which, along with their homologs, are prime candidates to specifically drive gene expression during meiosis in plants and can thus provide important tools for meiosis study and crop breeding.