RESUMO
Meiotic homologous chromosomes synapse and undergo crossing over (CO). In many eukaryotes, both synapsis and crossing over require the induction of double stranded breaks (DSBs) and subsequent repair via homologous recombination. In these organisms, two key proteins are recombinases RAD51 and DMC1. Recombinase-modulators HOP2 and MND1 assist RAD51 and DMC1 and also are required for synapsis and CO. We have investigated the hop2-1 phenotype in Arabidopsis during the segregation stages of both meiosis and mitosis. Despite a general lack of synapsis during prophase I, we observed extensive, stable interconnections between nonhomologous chromosomes in diploid hop2-1 nuclei in first and second meiotic divisions. Using γH2Ax as a marker of unrepaired DSBs, we detected γH2AX foci from leptotene through early pachytene but saw no foci from mid-pachytene onward. We conclude that the bridges seen from metaphase I onward are due to mis-repaired DSBs, not unrepaired ones. Examining haploids, we found that wild type haploids produce only univalents, but hop2-1 haploids like hop2-1 diploids have illegitimate connections stable enough to produce bridged chromosomes during segregation. Our results suggest that HOP2 has a significant active role in preventing repairs that use nonhomologous chromosomes during meiosis. We also found evidence that HOP2 plays a role in preventing illegitimate repair of radiation-induced DSBs in rapidly dividing petal cells. We conclude that HOP2 in Arabidopsis plays both a positive role in promoting synapsis and a separable role in preventing DSB repair using nonhomologous chromosomes. SIGNIFICANCE STATEMENT : The fidelity of homologous recombination (HR) during meiosis is essential to the production of viable gametes and for maintaining genome integrity in vegetative cells. HOP2 is an important protein for accurate meiotic HR in plants. We have found evidence of high levels of illegitimate repairs between nonhomologous chromosomes during meiosis and in irradiated petal cells in hop2-1 mutants, suggesting a role for HOP2 beyond its established role in synapsis and crossing over.
Assuntos
Arabidopsis , Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Pareamento Cromossômico , Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Rad51 Recombinase/genéticaRESUMO
Meristem function is underpinned by numerous genes that affect hormone levels, ultimately controlling phyllotaxy, the transition to flowering and general growth properties. Class I KNOX genes are major contributors to this process, promoting cytokinin biosynthesis but repressing gibberellin production to condition a replication competent state. We identified a suppressor mutant of the KNOX1 mutant brevipedicellus (bp) that we termed flasher (fsh), which promotes stem and pedicel elongation, suppresses early senescence, and negatively affects reproductive development. Map-based cloning and complementation tests revealed that fsh is due to an E40K change in the flavin monooxygenase GS-OX5, a gene encoding a glucosinolate (GSL) modifying enzyme. In vitro enzymatic assays revealed that fsh poorly converts substrate to product, yet the levels of several GSLs are higher in the suppressor line, implicating FSH in feedback control of GSL flux. FSH is expressed predominantly in the vasculature in patterns that do not significantly overlap those of BP, implying a non-cell autonomous mode of meristem control via one or more GSL metabolites. Hormone analyses revealed that cytokinin levels are low in bp, but fsh restores cytokinin levels to near normal by activating cytokinin biosynthesis genes. In addition, jasmonate levels in the fsh suppressor are significantly lower than in bp, which is likely due to elevated expression of JA inactivating genes. These observations suggest the involvement of the GSL pathway in generating one or more negative effectors of growth that influence inflorescence architecture and fecundity by altering the balance of hormonal regulators.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Glucosinolatos/metabolismo , Proteínas de Homeodomínio/genética , Reguladores de Crescimento de Plantas/fisiologia , Proteínas de Plantas/genética , Arabidopsis/anatomia & histologia , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Flores/anatomia & histologia , Flores/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Homeostase/genética , Mutação/genética , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologiaRESUMO
Mutations in the BREVIPEDICELLUS (BP) gene of Arabidopsis thaliana condition a pleiotropic phenotype featuring defects in internode elongation, the homeotic conversion of internode to node tissue, and downward pointing flowers and pedicels. We have characterized five mutant alleles of BP, generated by EMS, fast neutrons, x-rays, and aberrant T-DNA insertion events. Curiously, all of these mutagens resulted in large deletions that range from 140 kbp to over 900 kbp just south of the centromere of chromosome 4. The breakpoints of these mutants were identified by employing inverse PCR and DNA sequencing. The south breakpoints of all alleles cluster in BAC T12G13, while the north breakpoint locations are scattered. With the exception of a microhomology at the bp-5 breakpoint, there is no homology in the junction regions, suggesting that double-stranded breaks are repaired via non-homologous end joining. Southwestern blotting demonstrated the presence of nuclear matrix binding sites in the south breakpoint cluster (SBC), which is A/T rich and possesses a variety of repeat sequences. In situ hybridization on pachytene chromosome spreads complemented the molecular analyses and revealed heretofore unrecognized structural variation between the Columbia and Landsberg erecta genomes. Data mining was employed to localize other large deletions around the HY4 locus to the SBC region and to show that chromatin modifications in the region shift from a heterochromatic to euchromatic profile. Comparisons between the BP/HY4 regions of A. lyrata and A. thaliana revealed that several chromosome rearrangement events have occurred during the evolution of these two genomes. Collectively, the features of the region are strikingly similar to the features of characterized metazoan chromosome fragile sites, some of which are associated with karyotype evolution.
Assuntos
Arabidopsis , Aberrações Cromossômicas , Sítios Frágeis do Cromossomo/genética , Regiões de Interação com a Matriz/genética , Arabidopsis/citologia , Arabidopsis/genética , Evolução Biológica , Centrômero/genética , Aberrações Cromossômicas/efeitos dos fármacos , Aberrações Cromossômicas/efeitos da radiação , Quebra Cromossômica , DNA Bacteriano , Flores/efeitos dos fármacos , Flores/genética , Flores/efeitos da radiação , Deleção de Genes , Mutagênese Insercional , Mutação/efeitos dos fármacos , Mutação/efeitos da radiação , Nêutrons , Raios XRESUMO
HOP2 is a conserved protein that plays a positive role in homologous chromosome pairing and a separable role in preventing illegitimate connections between nonhomologous chromosome regions during meiosis. We employed ChIP-seq to discover that Arabidopsis HOP2 binds along the length of all chromosomes, except for centromeric and nucleolar organizer regions, and no binding sites were detected in the organelle genomes. A large number of reads were assigned to the HOP2 locus itself, yet TAIL-PCR and SNP analysis of the aligned sequences indicate that many of these reads originate from the transforming T-DNA, supporting the role of HOP2 in preventing nonhomologous exchanges. The 292 ChIP-seq peaks are largely found in promoter regions and downstream from genes, paralleling the distribution of recombination hotspots, and motif analysis revealed that there are several conserved sequences that are also enriched at crossover sites. We conducted coimmunoprecipitation of HOP2 followed by LC-MS/MS and found enrichment for several proteins, including some histone variants and modifications that are also known to be associated with recombination hotspots. We propose that HOP2 may be directed to chromatin motifs near double strand breaks, where homology checks are proposed to occur.
RESUMO
We previously developed an online multiple-choice question authoring, learning, and self-assessment tool that we termed Quizzical. Here we report statistical analyses over two consecutive years of Quizzical use in a large sophomore-level introductory molecular biology course. Students were required to author two questions during the term and were also afforded opportunities to earn marks for quiz participation. We found that students whose final grade was "A," "B," or "C" exhibited similar patterns of Quizzical engagement. The degree to which students participated was positively associated with performance on formal exams, even if prior academic performance was considered as a covariable. During both terms investigated, students whose Quizzical engagement increased from one exam to the next earned statistically significant higher scores on the subsequent exam, and students who attempted Quizzical questions from earlier in the term scored higher, on average, on the cumulative portion of the final exam. We conclude that the structure and value of the assignment, and the utility of Quizzical as a discipline-independent active-learning and self-assessment tool, enabled students to better master course topics.
Assuntos
Desempenho Acadêmico , Avaliação Educacional , Humanos , Aprendizagem , Aprendizagem Baseada em Problemas , EstudantesRESUMO
BREVIPEDICELLUS (BP) encodes a class-I KNOTTED1-like homeobox (KNOX) transcription factor that plays a critical role in conditioning a replication competent state in the apical meristem, and it also governs growth and cellular differentiation in internodes and pedicels. To search for factors that modify BP signaling, we conducted a suppressor screen on bp er (erecta) plants and identified a mutant that ameliorates many of the pleiotropic defects of the parent line. Map based cloning and complementation studies revealed that the defect lies in the FILAMENTOUS FLOWER (FIL) gene, a member of the YABBY family of transcriptional regulators that contribute to meristem organization and function, phyllotaxy, leaf and floral organ growth and polarity, and are also known to repress KNOX gene expression. Genetic and cytological analyses of the fil-10 suppressor line indicate that the role of FIL in promoting growth is independent of its previously characterized influences on meristem identity and lateral organ polarity, and likely occurs non-cell-autonomously from superior floral organs. Transcription profiling of inflorescences revealed that FIL downregulates numerous transcription factors which in turn may subordinately regulate inflorescence architecture. In addition, FIL, directly or indirectly, activates over a dozen genes involved in glucosinolate production in part by activating MYB28, a known activator of many aliphatic glucosinolate biosynthesis genes. In the bp er fil-10 suppressor mutant background, enhanced expression of CYP71A13, AMIDASE1 (AMI) and NITRILASE genes suggest that auxin levels can be modulated by shunting glucosinolate metabolites into the IAA biosynthetic pathway, and increased IAA levels in the bp er fil-10 suppressor accompany enhanced internode and pedicel elongation. We propose that FIL acts to oppose KNOX1 gene function through a complex regulatory network that involves changes in secondary metabolites and auxin.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Glucosinolatos/metabolismo , Proteínas de Homeodomínio/genética , Ácidos Indolacéticos/metabolismo , Mutação , Proteínas de Plantas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Glucosinolatos/genética , Proteínas de Homeodomínio/metabolismo , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Fenótipo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Plant seeds naturally accumulate storage reserves (proteins, carbohydrates, lipids) that are mobilized during germination to provide energy and raw materials to support early seedling growth. Seeds have been exploited as bioreactors for the production to foreign materials, but stable, high level expression has been elusive, in part due to the intrinsic bias for producing the natural reserves in their typical proportions. To identify mutants governing seed filling, we screened a population of mutagenized Arabidopsis plants for a mutant that failed to fill its seeds. Here we report the identification of ssp1, a recessive, viable mutant that accumulates approximately 15% less protein than wildtype seeds. Molecular analyses revealed that ssp1 is due to the introduction of a premature stop codon in CRU3, one of the major cruciferin genes. Unlike many other reserve mutants or transgenic lines in which seed storage protein levels are reduced by antisense/RNAi technologies, ssp1 exhibits low level compensation by other reserves, and represents a mutant background that might prove useful for high level expression of foreign proteins. To test this hypothesis, we used a bean phytohemagglutinin (PHA) gene as a reporter and compared PHA expression levels in single copy insertion lines in ssp1 vs. wildtype. These near isogenic lines allow reporter protein levels to be compared without the confounding and sometimes unknown influences of transgene copy number and position effects on gene expression. The ssp1 lines consistently accumulated more PHA than the backcrossed counterparts, with increases ranging from 12% to 126%. This proof of principle study suggests that similar strategies in crop plants may improve the yield of foreign proteins of agronomic and economic interest.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Expressão Gênica , Mutação , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Metabolismo dos Lipídeos , Fenótipo , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/metabolismoRESUMO
In higher eukaryotes, the condensin complex is a multisubunit apparatus that plays a pivotal role in the coordinated condensation of chromatin during mitosis. The catalytic subunits, CAP-E and CAP-C, members of the SMC family of ATPases, form a heterodimer, the activity of which is controlled by the non-SMC subunits CAP-D2, CAP-G and CAP-H. Here, we report the characterization of a T-DNA insertion mutant of the Arabidopsis CAP-C gene. Analysis of the progeny of selfed heterozygotes revealed that the homozygous null genotype is embryo lethal, with arrest occurring at or before the globular stage of development. Patterning defects associated with altered planes of cytokinesis were found in both the embryo and the suspensor. Crosses of heterozygotes with wild type plants revealed both male and female gametophytic defects. Stretched chromatin was observed between segregating mitotic chromosomes in pollen produced by selfed heterozygotes. Additionally, some plants heterozygous for the T-DNA insertion exhibited loss of apical dominance and mild fasciation, indicating a semi-dominant effect of the mutation. These results reveal a critical role for AtCAP-C during cell division and, unlike our previous studies on the AtCAP-E genes, suggest that no redundant factors for AtCAP-C exist in the Arabidopsis genome.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Proteínas de Ligação a DNA/fisiologia , Desenvolvimento Embrionário/fisiologia , Gametogênese/fisiologia , Complexos Multiproteicos/fisiologia , Adenosina Trifosfatases/genética , Arabidopsis/embriologia , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica , Genes de Plantas , Heterozigoto , Família Multigênica , Complexos Multiproteicos/genética , Mutação , Fenótipo , Pólen/crescimento & desenvolvimentoRESUMO
Although the regulation of Arabidopsis floral meristem patterning and determinacy has been studied in detail, very little is known about the genetic mechanisms directing development of the pedicel, the short stem linking the flower to the inflorescence axis. Here, we provide evidence that the pedicel consists of a proximal portion derived from the young flower primordium, and a bulged distal region that emerges from tissue at the bases of sepals in the floral bud. Distal pedicel growth is controlled by the KNOTTED1-like homeobox gene BREVIPEDICELLUS (BP), as 35S::BP plants show excessive proliferation of pedicel tissue, while loss of BP conditions a radial constriction around the distal pedicel circumference. Mutant radial constrictions project proximally along abaxial and lateral sides of pedicels, leading to occasional downward bending at the distal pedicel. This effect is severely enhanced in a loss-of-function erecta (er) background, resulting in radially constricted tissue along the entire abaxial side of pedicels and downward-oriented flowers and fruit. Analysis of pedicel vascular patterns revealed biasing of vasculature towards the abaxial side, consistent with a role for BP and ER in regulating a vascular-borne growth inhibitory signal. BP expression in a reporter line marked boundaries between the inflorescence stem and lateral organs and the receptacle and floral organs. This boundary expression appears to be important to prevent homeotic displacement of node and lateral organ fates into underlying stem tissue. To investigate interactions between pedicel and flower development, we crossed bp er into various floral mutant backgrounds. Formation of laterally-oriented bends in bp lfy er pedicels paralleled phyllotaxy changes, consistent with a model where the architecture of mutant stems is controlled by both organ positioning and vasculature patterns. Collectively, our results indicate that the BP gene acts in Arabidopsis stems to confer a growth-competent state that counteracts lateral-organ associated asymmetries and effectively radializes internode and pedicel growth and differentiation patterns.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Genes Homeobox , Genes de Plantas , Caules de Planta/genética , Cruzamentos Genéticos , Regulação da Expressão Gênica de Plantas , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Meristema , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Modelos Biológicos , Morfogênese , Mosaicismo , Mutação , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Transcrição GênicaRESUMO
Plant architecture is dictated by morphogenetic factors that specify the number and symmetry of lateral organs as well as their positions relative to the primary axis. Mutants defective in the patterning of leaves and floral organs have provided new insights on the signaling pathways involved, but there is comparatively little information regarding aspects of the patterning of stems, which play a dominant role in architecture. To this end, we have characterized five alleles of the brevipedicellus mutant of Arabidopsis, which exhibits reduced internode and pedicel lengths, bends at nodes, and downward-oriented flowers and siliques. Bends in stems correlate with a loss of chlorenchyma tissue at the node adjacent to lateral organs and in the abaxial regions of pedicels. A stripe of achlorophyllous tissue extends basipetally from each node and is positioned over the vasculature that services the corresponding lateral organ. Map-based cloning and complementation studies revealed that a null mutation in the KNAT1 homeobox gene is responsible for these pleiotropic phenotypes. Our observation that wild-type Arabidopsis plants also downregulate chlorenchyma development adjacent to lateral organs leads us to propose that KNAT1 and ERECTA are required to restrict the action of an asymmetrically localized, vasculature-associated chlorenchyma repressor at the nodes. Our data indicate that it is feasible to alter the architecture of ornamental and crop plants by manipulating these genetically defined pathways.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Homeodomínio/genética , Proteínas de Plantas/genética , Caules de Planta/genética , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Alelos , Arabidopsis/crescimento & desenvolvimento , Divisão Celular/genética , Mapeamento Cromossômico/métodos , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Meristema/genética , Meristema/crescimento & desenvolvimento , Microscopia Eletrônica de Varredura , Mutação , Fenótipo , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/ultraestrutura , Transdução de Sinais/genéticaRESUMO
Proper chromatin condensation and sister chromatid resolution are essential for the maintenance of chromosomal integrity during cell division, and is in part mediated by a conserved multisubunit apparatus termed the condensin complex. The core subunits of the complex are members of the SMC2 (Structural Maintenance of Chromosomes) and SMC4 gene families. We have cloned an Arabidopsis gene, AtCAP-E1, which is a functional ortholog of the yeast SMC2 gene. A second, highly homologous SMC2 gene, AtCAPE-2, was identified by the Arabidopsis genome project. SMC2 gene expression in Arabidopsis was correlated with the mitotic activity of tissues, with high level expression observed in meristematic cells. The two genes are differentially expressed with AtCAP-E1 accounting for more than 85% of the total SMC2 transcript pool. The titan3 mutant is the result of a T-DNA insertion into AtCAP-E1, but other than subtle endosperm defects, titan3 is viable and fecund. We identified a T-DNA insertion mutant of AtCAP-E2, which showed no obvious mutant phenotype, indicating that the two genes are functionally redundant. Genetic crosses were employed to examine the consequences of reduced SMC2 levels. Both male and female gametogenesis were compromised in double mutant spores. Embryo lethality was observed for both double homozygous and AtCAP-E1(-/-), AtCAP-E2(+/-) plants; arrest occurred at or before the globular stage and was associated with altered planes of cell division in both the suspensor and the embryo. Down regulation of both genes by antisense technology, as well as in AtCAP-E1(+/-), AtCAP-E2(-/-) plants results in meristem disorganization and fasciation. Our data are consistent with the interpretation that threshold levels of SMC2 proteins are required for normal development and that AtCAP-E2 may have a higher affinity for its target than AtCAP-E1.