Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement.
BACKGROUND For over a century, genetic diversity of wheat worldwide was eroded by continual selection for high yields and industrial demands. Wheat landraces cultivated in Israel and Palestine demonstrate high genetic diversity and a potentially wide repertoire of adaptive alleles. While most Israeli-Palestinian wheat landraces were lost in the transition to 'Green Revolution' semi-dwarf varieties, some germplasm collections made at the beginning of the 20th century survived in gene banks and private collections worldwide. However, fragmentation and poor conservation place this unique genetic resource at a high risk of genetic erosion. Herein, we describe a long-term initiative to restore, conserve, and characterize a collection of Israeli and Palestinian wheat landraces (IPLR). RESULTS We report on (i) the IPLR construction (n = 932), (ii) the historical and agronomic context to this collection, (iii) the characterization and assessment of the IPLR's genetic diversity, and (iv) a data comparison from two distinct subcollections within IPLR: a collection made by N. Vavilov in 1926 (IPLR-VIR) and a later one (1979-1981) made by Y. Mattatia (IPLR-M). Though conducted in the same eco-geographic space, these two collections were subjected to considerably different conservation pathways. IPLR-M, which underwent only one propagation cycle, demonstrated marked genetic and phenotypic variability (within and between accessions) in comparison with IPLR-VIR, which had been regularly regenerated over similar to 90 years. CONCLUSION We postulate that long-term ex situ conservation involving human and genotype x environment selection may significantly reduce accession heterogeneity and allelic diversity. Results are further discussed in a broader context of pre-breeding and conservation. (c) 2019 Society of Chemical Industry
AtRad52 homologs are involved in DNA recombination and repair, but their precise functions in different homologous recombination (HR) pathways or in gene-targeting have not been analyzed. In order to facilitate our analyses, we generated an AtRad52-1A variant that had a stronger nuclear localization than the native gene thanks to the removal of the transit peptide for mitochondrial localization and to the addition of a nuclear localization signal. Over-expression of this variant increased HR in the nucleus, compared with the native AtRad52-1A: it increased intra-chromosomal recombination and synthesis-dependent strand-annealing HR repair rates; but conversely, it repressed the single-strand annealing pathway. The effect of AtRad52-1A over-expression on gene-targeting was tested with and without the expression of small RNAs generated from an RNAi construct containing homology to the target and donor sequences. True gene-targeting events at the Arabidopsis Cruciferin locus were obtained only when combining AtRad52-1A over-expression and target/donor-specific RNAi. This suggests that sequence-specific small RNAs might be involved in AtRad52-1A-mediated HR.
Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon, we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron-induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.
Agrobacterium tumefaciens mediated T-DNA integration is a common tool for plant genome manipulation. However, there is controversy regarding whether T-DNA integration is biased towards genes or randomly distributed throughout the genome. In order to address this question, we performed high-throughput mapping of T-DNA-genome junctions obtained in the absence of selection at several time points after infection. T-DNA-genome junctions were detected as early as 6 hours post-infection. T-DNA distribution was apparently uniform throughout the chromosomes, yet local biases toward AT-rich motifs and T-DNA border sequence micro-homology were detected. Analysis of the epigenetic landscape of previously isolated sites of T-DNA integration in Kanamycin-selected transgenic plants showed an association with extremely low methylation and nucleosome occupancy. Conversely, non-selected junctions from this study showed no correlation with methylation and had chromatin marks, such as high nucleosome occupancy and high H3K27me3, that correspond to three-dimensional-interacting heterochromatin islands embedded within euchromatin. Such structures may play a role in capturing and silencing invading T-DNA.
Background: The merging of genomes in inter-specific hybrids can result in novel phenotypes, including increased growth rate and biomass yield, a phenomenon known as heterosis. Heterosis is typically viewed as the opposite of hybrid incompatibility. In this view, the superior performance of the hybrid is attributed to heterozygote combinations that compensate for deleterious mutations accumulating in each individual genome, or lead to new, over-dominating interactions with improved performance. Still, only fragmented knowledge is available on genes and processes contributing to heterosis.Results: We describe a budding yeast hybrid that grows faster than both its parents under different environments. Phenotypically, the hybrid progresses more rapidly through cell cycle checkpoints, relieves the repression of respiration in fast growing conditions, does not slow down its growth when presented with ethanol stress, and shows increased signs of DNA damage. A systematic genetic screen identified hundreds of S. cerevisiae alleles whose deletion reduced growth of the hybrid. These growth-affecting alleles were condition-dependent, and differed greatly from alleles that reduced the growth of the S. cerevisiae parent.Conclusions: Our results define a budding yeast hybrid that is perturbed in multiple regulatory processes but still shows a clear growth heterosis. We propose that heterosis results from incompatibilities that perturb regulatory mechanisms, which evolved to protect cells against damage or prepare them for future challenges by limiting cell growth.
Bread is consumed daily by billions of people, yet evidence regarding its clinical effects is contradicting. Here, we performed a randomized crossover trial of two 1-week-long dietary interventions comprising consumption of either traditionally made sourdough-leavened whole-grain bread or industrially made white bread. We found no significant differential effects of bread type on multiple clinical parameters. The gut microbiota composition remained person specific throughout this trial and was generally resilient to the intervention. We demonstrate statistically significant interpersonal variability in the glycemic response to different bread types, suggesting that the lack of phenotypic difference between the bread types stems from a person-specific effect. We further show that the type of bread that induces the lower glycemic response in each person can be predicted based solely on microbiome data prior to the intervention. Together, we present marked personalization in both bread metabolism and the gut microbiome, suggesting that understanding dietary effects requires integration of person-specific factors.
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.
Homologous recombination affects genome evolution through crossover, gene conversion and point mutations. Whole genome sequencing together with a detailed epigenome analysis have shed new light on our understanding of how meiotic recombination shapes plant genes and genome structure. Crossover events are associated with DNA sequence motifs, together with an open chromatin signature (hypomethylated CpGs, low nucleosome occupancy or specific histone modifications). The crossover landscape may differ between male and female meiocytes and between species. At the gene level, crossovers occur preferentially in promoter regions in Arabidopsis. In recent years, there is rising support suggesting that biased mismatch repair during meiotic recombination may increase GC content genome-wide and may be responsible for the GC content gradient found in many plant genes.
Wheat straw is a potential source of feedstock for biofuel production that does not compete with food. We have screened 48 wheat lines from a collection representing a broad range of the biodiversity of wild and domestic wheat. Wheat straw was fractionated into water-soluble and nonsoluble fractions. In the water-soluble fraction (WSF), we found a broad variation in the concentration of free soluble sugars (FSS) and a narrow variation in starch. The FSS fraction could reach levels of reducing sugars as high as 130 g sugar/kg of straw. The analysis of the FSS by ion chromatography indicated that fructose and glucose were the major sugar monomers in this fraction. The composition of the nonsoluble cell wall fraction was determined by both pyrolysis and direct chemical analysis. These analyses showed a limited variation in the lignin or the cellulose fraction. There was a significant degree of variation among wheat lines in the enzymatic saccharification of the straw, following acid pretreatment. Interestingly, the straw from wild wheat had the highest degree of saccharification compared to domestic lines. These findings are of interest for the biofuel industry because they mean that wheat lines can be developed in which a significant amount of free soluble sugars can be easily extracted from straw without the need for costly pretreatment and enzymatic deconstruction. Moreover, the high FSS trait might be combined with the high enzymatic saccharification trait suggesting that wheat lines can be developed with a straw composition better adapted for biofuel production.
2015 American Society of Plant Biologists. All rights reserved.The rate of crossover, the reciprocal exchanges of homologous chromosomal segments, is not uniform along chromosomes differing between male and female meiocytes. To better understand the factors regulating this variable landscape, we performed a detailed genetic and epigenetic analysis of 737 crossover events in Arabidopsis thaliana. Crossovers were more frequent than expected in promoters. Three DNA motifs enriched in crossover regions and less abundant in crossover-poor pericentric regions were identified. One of these motifs, the CCN repeat, was previously unknown in plants. The A-rich motif was preferentially associated with promoters, while the CCN repeat and the CTT repeat motifs were preferentially associated with genes. Analysis of epigenetic modifications around the motifs showed, in most cases, a specific epigenetic architecture. For example, we show that there is a peak of nucleosome occupancy and of H3K4me3 around the CCN and CTT repeat motifs while nucleosome occupancy was lowest around the A-rich motif. Cytosine methylation levels showed a gradual decrease within 2 kb of the three motifs, being lowest at sites where crossover occurred. This landscape was conserved in the decreased DNA methylation1 mutant. In summary, the crossover motifs are associated with epigenetic landscapes corresponding to open chromatin and contributing to the nonuniformity of crossovers in Arabidopsis.
Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.
We speculate that multicopy transposons, carrying both fitness and unfitness genes, can provide new positive and negative selection options to intractable weed problems. Multicopy transposons rapidly disseminate through populations, appearing in approximately 100% of progeny, unlike nuclear transgenes, which appear in a proportion of segregating populations. Different unfitness transgenes and modes of propagation will be appropriate for different cases: (1) outcrossing Amaranthus spp. (that evolved resistances to major herbicides); (2) Lolium spp., important pasture grasses, yet herbicide-resistant weeds in crops; (3) rice (Oryza sativa), often infested with feral weedy rice, which interbreeds with the crop; and (4) self-compatible sorghum (Sorghum bicolor), which readily crosses with conspecific shattercane and with allotetraploid johnsongrass (Sorghum halepense). The speculated outcome of these scenarios is to generate weed populations that contain the unfitness gene and thus are easily controllable. Unfitness genes can be under chemically or environmentally inducible promoters, activated after gene dissemination, or under constitutive promoters where the gene function is utilized only at special times (e. g. sensitivity to an herbicide). The transposons can be vectored to the weeds by introgression from the crop (in rice, sorghum, and Lolium spp.) or from planted engineered weed (Amaranthus spp.) using a gene conferring the degradation of a no longer widely used herbicide, especially in tandem with an herbicide-resistant gene that kills all nonhybrids, facilitating the rapid dissemination of the multicopy transposons in a weedy population.
Gene Targeting (GT) is the integration of an introduced vector into a specific chromosomal site, via homologous recombination. It is considered an effective tool for precise genome editing, with far-reaching implications in biological research and biotechnology, and is widely used in mice, with the potential of becoming routine in many species. Nevertheless, the epigenetic status of the targeted allele remains largely unexplored. Using GT-modified lines of the model plant Arabidopsis thaliana, we show that the DNA methylation profile of the targeted locus is changed following GT. This effect is non-directional as methylation can be either completely lost, maintained with minor alterations or show instability in the generations subsequent to GT. As DNA methylation is known to be involved in several cellular processes, GT-related alterations may result in unexpected or even unnoticed perturbations. Our analysis shows that GT may be used as a new tool for generating epialleles, for example, to study the role of gene body methylation. In addition, the analysis of DNA methylation at the targeted locus may be utilized to investigate the mechanism of GT, many aspects of which are still unknown.
Custom-designed nucleases are a promising technology for genome editing through the catalysis of double-strand DNA breaks within target loci and subsequent repair by the host cell, which can result in targeted mutagenesis or gene replacement. Implementing this new technology requires a rapid means to determine the cleavage efficiency of these custom-designed proteins in planta. Here we present such an assay that is based on cleavage-dependent luciferase gene correction as part of a transient dual-luciferase(A (R)) reporter (Promega) expression system. This assay consists of co-infiltrating Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains: one contains the target sequence embedded within a luciferase reporter gene and the second strain contains the custom-designed nuclease gene(s). We compared repair following site-specific nuclease digestion through non-homologous DNA end-joining, as opposed to single strand DNA annealing, as a means to restore an out-of-frame luciferase gene cleavage-reporter construct. We show, using luminometer measurements and bioluminescence imaging, that the assay for non-homologous end-joining is sensitive, quantitative, reproducible and rapid in estimating custom-designed nucleases' cleavage efficiency. We detected cleavage by two out of three transcription activator-like effector nucleases that we custom-designed for targets in the Arabidopsis CRUCIFERIN3 gene, and we compared with the well-established 'QQR' zinc-finger nuclease. The assay we report requires only standard equipment and basic plant molecular biology techniques, and it can be carried out within a few days. Different types of custom-designed nucleases can be preliminarily tested in our assay system before their downstream application in plant genome editing.
The evolvement of duplicated gene loci in allopolyploid plants has become the subject of intensive studies. Most duplicated genes remain active in neoallopolyploids contributing either to a favourable effect of an extra gene dosage or to the build-up of positive inter-genomic interactions when genes or regulation factors on homoeologous chromosomes are divergent. However, in a small number of loci (about 10%), genes of only one genome are active, while the homoeoalleles on the other genome(s) are either eliminated or partially or completely suppressed by genetic or epigenetic means. For several traits, the retention of controlling genes is not random, favouring one genome over the other(s). Such genomic asymmetry is manifested in allopolyploid wheat by the control of various morphological and agronomical traits, in the production of rRNA and storage proteins, and in interaction with pathogens. It is suggested that the process of cytological diploidization leading to exclusive intra-genomic meiotic pairing and, consequently, to complete avoidance of inter-genomic recombination, has two contrasting effects. Firstly, it provides a means for the fixation of positive heterotic inter-genomic interactions and also maintains genomic asymmetry resulting from loss or silencing of genes. The possible mechanisms and evolutionary advantages of genomic asymmetry are discussed.
Meiotic recombination is tightly regulated by cis- and trans-acting factors. Although DNA methylation and chromatin remodeling affect chromosome structure, their impact on meiotic recombination is not well understood. To study the effect of DNA methylation on the landscape of chromosomal recombination, we analyzed meiotic recombination in the decreased DNA methylation 1 (ddm1) mutant. DDM1 is a SWI2/SNF2-like chromatin-remodeling protein necessary for DNA methylation and heterochromatinmaintenance in Arabidopsis thaliana. The rate of meiotic recombination between markers located in euchromatic regions was significantly higher in both heterozygous (DDM1/ddm1) and homozygous (ddm1/ddm1) backgrounds than in WT plants. The effect on recombination was similar for both male and female meiocytes. Contrary to expectations, ddm1 had no effect on the number of crossovers between markers in heterochromatic pericentric regions that underwent demethylation. These results are surprising, because the pericentromeric regions are hypermethylated and were expected to be the regions most affected by demethylation. Thus, DDM1 loss of function may trigger changes that enhance meiotic recombination in euchromatin regions but are not sufficient to induce the same events in heterochromatic segments. This work uncovers the repressive role of methylation on meiotic recombination in euchromatic regions and suggests that additional factors may have a role in controlling the suppression of recombination in heterochromatin.
The wheat group has evolved through allopolyploidization, namely, through hybridization among species from the plant genera Aegilops and Triticum followed by genome doubling. This speciation process has been associated with ecogeographical expansion and with domestication. In the past few decades, we have searched for explanations for this impressive success. Our studies attempted to probe the bases for the wide genetic variation characterizing these species, which accounts for their great adaptability and colonizing ability. Central to our work was the investigation of how allopolyploidization alters genome structure and expression. We found in wheat that allopolyploidy accelerated genome evolution in two ways: (1) it triggered rapid genome alterations through the instantaneous generation of a variety of cardinal genetic and epigenetic changes (which we termed "revolutionary" changes), and (2) it facilitated sporadic genomic changes throughout the species' evolution (i.e., evolutionary changes), which are not attainable at the diploid level. Our major findings in natural and synthetic allopolyploid wheat indicate that these alterations have led to the cytological and genetic diploidization of the allopolyploids. These genetic and epigenetic changes reflect the dynamic structural and functional plasticity of the allopolyploid wheat genome. The significance of this plasticity for the successful establishment of wheat allopolyploids, in nature and under domestication, is discussed.
Targeted modification of the genome is an important genetic tool, which can be achieved via homologous, non-homologous or site-specific recombination. Although numerous efforts have been made, such a tool does not exist for routine applications in plants. This work describes a simple and useful method for targeted mutagenesis or gene targeting, tailored to floral-dip transformation in Arabidopsis, by means of specific protein expression in the egg cell. Proteins stably or transiently expressed under the egg apparatus-specific enhancer (EASE) were successfully localized to the area of the egg cell. Moreover, a zinc-finger nuclease expressed under EASE induced targeted mutagenesis. Mutations obtained under EASE control corresponded to genetically independent events that took place specifically in the germline. In addition, RAD54 expression under EASE led to an approximately 10-fold increase in gene targeting efficiency, when compared with wild-type plants. EASE-controlled gene expression provides a method for the precise engineering of the Arabidopsis genome through temporally and spatially controlled protein expression. This system can be implemented as a useful method for basic research in Arabidopsis, as well as in the optimization of tools for targeted genetic modifications in crop plants.
Speciation via interspecific or intergeneric hybridization and polyploidization triggers genomic responses involving genetic and epigenetic alterations. Such modifications may be induced by small RNAs, which affect key cellular processes, including gene expression, chromatin structure, cytosine methylation and transposable element (TE) activity. To date, the role of small RNAs in the context of wide hybridization and polyploidization has received little attention. In this work, we performed high-throughput sequencing of small RNAs of parental, intergeneric hybrid, and allopolyploid plants that mimic the genomic changes occurring during bread wheat speciation. We found that the percentage of small RNAs corresponding to miRNAs increased with ploidy level, while the percentage of siRNAs corresponding to TEs decreased. The abundance of most miRNA species was similar to midparent values in the hybrid, with some deviations, as seen in overrepresentation of miR168, in the allopolyploid. In contrast, the number of siRNAs corresponding to TEs strongly decreased upon allopolyploidization, but not upon hybridization. The reduction in corresponding siRNAs, together with decreased CpG methylation, as shown here for the Veju element, represent hallmarks of TE activation. TE-siRNA downregulation in the allopolyploid may contribute to genome destabilization at the initial stages of speciation. This phenomenon is reminiscent of hybrid dysgenesis in Drosophila.
RADiation sensitive52 (RAD52) mediates RAD51 loading onto single-stranded DNA ends, thereby initiating homologous recombination and catalyzing DNA annealing. RAD52 is highly conserved among eukaryotes, including animals and fungi. This article reports that RAD52 homologs are present in all plants whose genomes have undergone extensive sequencing. Computational analyses suggest a very early RAD52 gene duplication, followed by later lineage-specific duplications, during the evolution of higher plants. Plant RAD52 proteins have high sequence similarity to the oligomerization and DNA binding N-terminal domain of RAD52 proteins. Remarkably, the two identified Arabidopsis thaliana RAD52 genes encode four open reading frames (ORFs) through differential splicing, each of which specifically localized to the nucleus, mitochondria, or chloroplast. The A. thaliana RAD52-1A ORF provided partial complementation to the yeast rad52 mutant. A. thaliana mutants and RNA interference lines defective in the expression of RAD52-1 or RAD52-2 showed reduced fertility, sensitivity to mitomycin C, and decreased levels of intrachromosomal recombination compared with the wild type. In summary, computational and experimental analyses provide clear evidence for the presence of functional RAD52 DNA-repair homologs in plants.
Background: Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination. Results: Using two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations. Conclusions: The evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence.
Homologous recombination (HR) is a central cellular process involved in many aspects of genome maintenance such as DNA repair, replication, telomere maintenance, and meiotic chromosomal segregation. HR is highly conserved among eukaryotes,contributing to genome stability as well as to the generation of genetic diversity. It has been intensively studied, for almost a century, in plants and in other organisms. In this antireview, rather than reviewing existing knowledge, we wish to underline the many open questions in plant HR. We will discuss the following issues: how do we define homology and how the degree of homology affects HR? Are there any plant-specific HR qualities, how extensive is functional conservation and did HR proteins acquire new functions? How efficient is HR in plants and what are the cis and the trans factors that regulate it? Finally, we will give the prospects for enhancing the rates of gene targeting and meiotic HR for plant breeding purposes.
In higher plants, the plastidial NADH dehydrogenase (Ndh) complex supports nonphotochemical electron fluxes from stromal electron donors to plastoquinones. Ndh functions in chloroplasts are not clearly established; however, its activity was linked to the prevention of the overreduction of stroma, especially under stress conditions. Here, we show by the characterization of Orr(Ds), a dominant transposon-tagged tomato (Solanum lycopersicum) mutant deficient in the NDH-M subunit, that this complex is also essential for the fruit ripening process. Alteration to the NDH complex in fruit changed the climacteric, ripening-associated metabolites and transcripts as well as fruit shelf life. Metabolic processes in chromoplasts of ripening tomato fruit were affected in Orr(Ds), as mutant fruit were yellow-orange and accumulated substantially less total carotenoids, mainly beta-carotene and lutein. The changes in carotenoids were largely influenced by environmental conditions and accompanied by modifications in levels of other fruit antioxidants, namely, flavonoids and tocopherols. In contrast with the pigmentation phenotype in mature mutant fruit, Orr(Ds) leaves and green fruits did not display a visible phenotype but exhibited reduced Ndh complex quantity and activity. This study therefore paves the way for further studies on the role of electron transport and redox reactions in the regulation of fruit ripening and its associated metabolism.
Organisms need genetic mechanisms to rapidly adapt to changing, stressful environments. Having a high mutation frequency would have a drag on a population due to the deleterious nature of mutations, but having a sub-population with high mutation rate due to the presence of mutator genes seems to be nature's solution. Far more is known about mutator genes in bacteria than in higher organisms. Mutator effects can be genetic, through mutations in genes that affect genome stability or it can be epigenetic through up- or down-regulation of these genes. The mutator genes can be genes with partially lost function, which deal with DNA replication or repair, or with detoxification of DNA-damaging cellular components. Transposons, which are sensitive to environmental stress, can also act as imitators in plants. Mutators can be constitutive or stress-induced. Most evidence for mutator-assisted evolution of stress resistance in plants is circumstantial, except for the evolution of atrazine herbicide resistance due to a nuclearly-inherited plastome mutator, which was repeated experimentally. An important feature of the mutator effect is that it is transient and is followed by reversion to the stable wild type, and can be counter-selected following outcrossing with the wild type. Similarly, "remembered" epigenetic stress-induced mutator effects were shown to last for a few generations. In summary, mutator genes could be playing an important role in the evolution of resistance to stress in plants, as it does in other systems, but to an extent that is yet unclear.
Allopolyploidy accelerates genome evolution in wheat in two ways: 1) allopolyploidization triggers rapid genome alterations (revolutionary changes) through the instantaneous generation of a variety of cardinal genetic and epigenetic changes, and 2) the allopolyploid condition facilitates sporadic genomic changes during the life of the species (evolutionary changes) that are not attainable at the diploid level. The revolutionary alterations, occurring during the formation of the allopolyploid and leading to rapid cytological and genetic diploidization, facilitate the successful establishment of the newly formed allopolyploid in nature. On the other hand, the evolutionary changes, occurring during the life of the allopolyploids, increase the intra-specific genetic diversity, and consequently, increased fitness, adaptability and competitiveness. These phenomena, emphasizing the dynamic plasticity of the allopolyploid wheat genome with regards to both structure and function, are described and discussed in this review.
Phytoliths are abundant in many archaeological sites, and can provide information on past vegetation. Very few analyses of their chemical composition have been made. Our measurements of the delta(13)C composition of modern wheat phytoliths suggest the presence of relatively large amounts of sugars and/or proteins in the water-soluble fraction, and lipids in the insoluble fraction. Other experimental approaches demonstrate that modern wheat phytoliths contain large quantities of glyco-conjugated proteins in a degraded state. One open question is whether or not phytoliths contain original DNA of the mother plant. Extracting protected ancient DNA from phytoliths would open many opportunities for progress in archaeobotanical studies. In order to address this question, we developed a method to dissolve phytoliths under conditions that do not degrade naked DNA, and showed that only a minimal amount of DNA was lost during the procedure. A hypersensitive assay did not, however, detect any DNA in extracts of phytoliths from an unburned phytolith-rich layer in Iron-Age sediments from Tel Dor, Israel. Extractions from modern phytolith samples of wheat also failed to provide any indication of DNA. We conclude that DNA is absent or not routinely recoverable in a random assembly of siliceous phytoliths. (C) 2007 Elsevier Ltd and INQUA. All rights reserved.
During evolution, novel phenotypes emerge through changes in gene expression, but the genetic basis is poorly understood. We compared the allele-specific expression of two yeast species and their hybrid, which allowed us to distinguish changes in regulatory sequences of the gene itself (cis) from changes in upstream regulatory factors (trans). Expression divergence between species was generally due to changes in cis. Divergence in trans reflected a differential response to the environment and explained the tendency of certain genes to diverge rapidly. Hybrid-specific expression, deviating from the parental range, occurred through novel cis-trans interactions or, more often, through modified trans regulation associated with environmental sensing. These results provide insights on the regulatory changes in cis and trans during the divergence of species and upon hybridization.
Starches extracted from most plant species are phosphorylated. alpha-Glucan water dikinase (GWD) is a key enzyme that controls the phosphate content of starch. In the absence of its activity starch degradation is impaired, leading to a starch excess phenotype in Arabidopsis and in potato leaves, and to reduced cold sweetening in potato tubers. Here, we characterized a transposon insertion (legwd::Ds) in the tomato GWD (LeGWD) gene that caused male gametophytic lethality. The mutant pollen had a starch excess phenotype that was associated with a reduction in pollen germination. SEM and TEM analyses indicated mild shrinking of the pollen grains and the accumulation of large starch granules inside the plastids. The level of soluble sugars was reduced by 1.8-fold in mutant pollen grains. Overall, the transmission of the mutant allele was only 0.4% in the male, whereas it was normal in the female. Additional mutant alleles, obtained through transposon excision, showed the same phenotypes as legwd::Ds. Moreover, pollen germination could be restored, and the starch excess phenotype could be abolished in lines expressing the potato GWD homolog (StGWD) under a pollen-specific promoter. In these lines, where fertility was restored, homozygous plants for legwd::Ds were isolated, and showed the starch excess phenotype in the leaves. Overall, our results demonstrate the importance of starch phosphorylation and breakdown for pollen germination, and open up the prospect for analyzing the role of starch metabolism in leaves and fruits.
The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rac154 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance severalfold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54 Delta mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.
Many plant roots acquire inorganic phosphate (Pi) from soils directly through the root-soil interface via high-affinity Pi transporters and/or through symbiotic associations between the cortical cells and arbuscular mycorrhizal fungi. In tomato, three phosphate transporters (LePT3, LePT4, and LePT5) are up-regulated upon colonization by arbuscular mycorrhizal fungi. In this study, the role of LePT4 in tomato is elucidated by molecular and physiological characterizations of a loss-of-function mutant lept4. In the absence of mycorrhizal infection and under solution-Pi concentrations (Cp) of 0.05 mM and 0.5 mM, the mutant exhibited severe Pi-deficiency symptoms which were associated with significantly lower Pi uptake as compared with that of the wild type. However, at a Cp of 5 mM, lept4 grew better than the wild type. Mycorrhizal infection at a Cp of 0.05 mM resulted in a significant increase in the transcripts of LePT4 in the wild type and a concomitant 2-fold increase in Pi uptake. Although upon mycorrhizal infection, lept4 also exhibited an increased Pi uptake, it was significantly lower than that of the wild type. Under a Cp of 1 mM and in the absence of mycorrhizal infection, LePT4 expression was suppressed in the wild type and a mutation in this gene resulted in a slight reduction in total Pi uptake. These data highlight the pivotal role of LePT4 in mycorrhizal-mediated Pi uptake in tomato, and show that this function may not be fully compensated by other members of the family. Characterization of the mycorrhiza-associated Pi transporter lept4 mutant, along with expression analysis of LePT3, provides evidence for the different routes of mycorrhiza-mediated Pi uptake in plants.
During homologous recombination (HR), a heteroduplex DNA is formed as a consequence of strand invasion. When the two homologous strands differ in sequence, a mismatch is generated. Earlier studies showed that mismatched heteroduplex often triggers abortion of recombination and that a pivotal component of this pathway is the mismatch repair Msh2 protein. In this study, we analysed the roles of AtMSH2 in suppression of recombination in Arabidopsis. We report that AtMSH2 has a broad range of anti-recombination effects: it suppresses recombination between divergent direct repeats in somatic cells or between homologues from different ecotypes during meiosis. This is the first example of a plant gene that affects HR as a function of sequence divergence and that has an anti-recombination meiotic effect. We discuss the implications of these results for plant improvement by gene transfer across species.
The floral homeotic APETALA3 (AP3) gene in Arabidopsis thaliana encodes a MADS box transcription factor required for specifying petal and stamen identities. AP3 is a member of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. Although Arabidopsis lacks genes in the paralogous Tomato MADS box gene 6 (TM6) lineage, tomato (Solanum lycopersicum) possesses both euAP3 and TM6 genes, which have functionally diversified. A loss- of-function mutation in Tomato AP3 (TAP3) resulted in homeotic transformations of both petals and stamens, whereas RNA interference induced reduction in TM6 function resulted in flowers with homeotic defects primarily in stamens. The functional differences between these genes can be ascribed partly to different expression domains. When overexpressed in an equivalent domain, both genes can partially rescue the tap3 mutant, indicating that relative levels as well as spatial patterns of expression contribute to functional differences. Our results also indicate that the two proteins have differing biochemical capabilities. Together, these results suggest that TM6 and TAP3 play qualitatively different roles in floral development; they also support the ideas that the ancestral role of AP3 lineage genes was in specifying stamen development and that duplication and divergence in the AP3 lineage allowed for the acquisition of a role in petal specification in the core eudicots.
The genome of plants, like that of other eukaryotes, is organized into chromatin, a compact structure that reduces the accessibility of DNA to machineries such as transcription, replication, and DNA recombination and repair. Plant genes, which contain the characteristic ATPase/helicase motifs of the chromatin remodeling Swi2/Snf2 family of proteins, have been thoroughly studied, but their role in homologous recombination or DNA repair has received limited attention. We have searched for homologs of the yeast RAD54 gene, whose role in recombination and repair and in chromatin remodeling is well established. Forty Arabidopsis SWI2/SNF2 genes were identified and the function of a selected group of 14 was analyzed. Mutant analysis and/or RNAi-mediated silencing showed that 11 of the 14 genes tested played a role in response to DNA damage. Two of the 14 genes were involved in homologous recombination between inverted repeats. The putative ortholog of RAD54 and close homologs of ERCC6/ RAD26 were involved in DNA damage response, Suggesting functional conservation across kingdoms. In addition, genes known for their role in development, such as PICKLE/GYMNOS and PIE1, or in silencing, Such as DDM1, turned Out to also be involved in DNA damage response. A comparison of ddm1 and met1 Mutants suggests that DNA damage response is affected essentially by chromatin structure and that cytosine methylation is less critical. These results emphasize the broad involvement of the SWI2/SNF2 family, and thus of chromatin remodeling, in genome maintenance and the link between epigenetic and genetic processes.
The olive tree (Olea europaea) was domesticated by vegetative propagation of selected wild individuals with Superior fruit. Later, new cultivars were established repeatedly from feral trees or from crosses between wild, feral, and domesticated trees. Thus the genetic background of many contemporary domesticated lines is a mixture of ancient cultivars and local wild trees. Ancient DNA may illuminate the complicated process of olive domestication because Such DNA sequences provide data about ancient genomes that existed closer to the domestication events. Well preserved DNA must be available for such studies, even though in the Mediterranean region, where olive cultivation took place, the climatic conditions are not favorable for DNA preservation. To select for well preserved pits we measured their proportions of lignin by IR spectroscopy. and correlated this with parameters of DNA quality such as template length in an olive-specific repeat array, and template quantity as determined by real-time PCR amplification. Archaeological pits that passed these tests did contain high quality ancient DNA. We present the first ancient olive DNA sequences and compare them to modern wild, feral and domesticated lines. (c) 2005 Elsevier Ltd. All rights reserved.
Gene targeting, which is homologous recombination-mediated integration of an extra-chromosomal DNA segment into a chromosomal target sequence, enables the precise disruption or replacement of any gene. Despite its value as a molecular genetic tool, gene targeting remains an inefficient technology in most species. We report that expression of the yeast RAD54 gene, a member of the SWI2/SNF2 chromatin remodeling gene family, enhances gene targeting in Arabidopsis by one to two orders of magnitude, from 10(-4) to 10(-3) in WT plants to 10(-2) to 10(-1). We show that integration events, detected with an assay based on the use of a fluorescent seed marker, are precise and germinally transmitted. These findings suggest that chromatin remodeling is rate-limiting for gene targeting in plants and improves the prospects for using gene targeting for the precise modification of plant genomes.
Solanaceous species are among the >200 000 plant species worldwide forming a mycorrhiza, that is, a root living in symbiosis with soil-borne arbuscular-mycorrhizal (AM) fungi. An important parameter of this symbiosis, which is vital for ecosystem productivity, agriculture, and horticulture, is the transfer of phosphate (Pi) from the AM fungus to the plant, facilitated by plasma membrane-spanning Pi transporter proteins. The first mycorrhiza-specific plant Pi transporter to be identified, was StPT3 from potato [Nature 414 (2004) 462]. Here, we describe novel Pi transporters from the solanaceous species tomato, LePT4, and its orthologue StPT4 from potato, both being members of the Pht1 family of plant Pi transporters. Phylogenetic tree analysis demonstrates clustering of both LePT4 and StPT4 with the mycorrhiza-specific Pi transporter from Medicago truncatula [Plant Cell, 14 (2002) 2413] and rice [Proc. Natl Acad Sci. USA, 99 (2002) 13324], respectively, but not with StPT3, indicating that two non-orthologous mycorrhiza-responsive genes encoding Pi transporters are co-expressed in the Solanaceae. The cloned promoter regions from both genes, LePT4 and StPT4, exhibit a high degree of sequence identity and were shown to direct expression exclusively in colonized cells when fused to the GUS reporter gene, in accordance with the abundance of LePT4 and StPT4 transcripts in mycorrhized roots. Furthermore, extensive sequencing of StPT4-like clones and subsequent expression analysis in potato and tomato revealed the presence of a close paralogue of StPT4 and LePT4, named StPT5 and LePT5, respectively, representing a third Pi transport system in solanaceous species which is upregulated upon AM fungal colonization of roots. Knock out of LePT4 in the tomato cv. MicroTom indicated considerable redundancy between LePT4 and other Pi transporters in tomato.
Meiotic recombination is a fundamental biological process that plays a central role in the evolution and breeding of plants. We have developed a new seed-based assay for meiotic recombination in Arabidopsis. The assay is based on the transformation of green and red fluorescent markers expressed under a seed-specific promoter. A total of 74 T-DNA markers were isolated, sequenced and mapped both physically and genetically. Lines containing red and green markers that map 1-20 cM apart were crossed to produce tester lines with the two markers linked in cis yielding seeds that fluoresced both in red and green. We show that these lines can be used for efficient scoring of recombinant types (red only or green only fluorescing seeds) in a seed population derived from a test cross (backcross) or self-pollination. Two tester lines that were characterized during several generations of backcross and self-pollination, one in the background of ecotype Landsberg and one in the ecotype Columbia, are described. We discuss the number of plants and seeds to be scored in order to obtain reliable and reproducible crossing over rate values. This assay offers a relatively high-throughput method, with the benefit of seed markers (similar to the maize classical genetic markers) combined with the advantages of Arabidopsis. It advances the prospect to better understand the factors that affect the rate of meiotic crossover in plants and to stimulate this process for more efficient breeding and mapping.
Keywords: COMMON WHEAT; CHROMOSOME LOCATION; TIBETAN WEEDRACE; GENE; MICROARRAYS; DNA
Recent studies have shown that allopolyploidy accelerates genome evolution in wheat in two ways: ( 1) allopolyploidization triggers rapid genome changes ( revolutionary changes) through the instantaneous generation of a variety of cardinal genetic and epigenetic alterations, and ( 2) the allopolyploid condition facilitates sporadic genomic changes during the life of the species ( evolutionary changes) that are not attainable at the diploid level. The revolutionary changes comprise ( 1) non-random elimination of coding and non-coding DNA sequences, ( 2) epigenetic changes such as DNA methylation of coding and non-coding DNA leading, among others, to gene silencing, ( 3) activation of genes and retroelements which in turn alters the expression of adjacent genes. These highly reproducible changes occur in the F-1 hybrids or in the first generation( s) of the nascent allopolyploids and were similar to those that occurred twice in nature: first in the formation of allotetraploid wheat (similar to 0.5 million years ago) and second in the formation of hexaploid wheat ( similar to 10,000 years ago). Elimination of non-coding sequences from one of the two homoeologous pairs in tetraploids and from two homoeologous pairs in hexaploids, augments the differentiation of homoeologous chromosomes at the polyploid level, thus providing the physical basis for the diploid-like meiotic behavior of allopolyploid wheat. Regulation of gene expression may lead to improved inter-genomic interactions. Gene inactivation brings about rapid diploidization while activation of genes through demethylation or through transcriptional activation of retroelements altering the expression of adjacent genes, leads to novel expression patterns. The evolutionary changes comprise ( 1) horizontal intergenomic transfer of chromosome segments between the constituent genomes, ( 2) production of recombinant genomes through hybridization and introgression between different allopolyploid species or, more s
The maize transposable element Activator (Ac) has been shown to be active in a number of dicots, including arabidopsis [Arabidopsis thaliana (L.) Heynh.], tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum Mill.), potato (Solanum tuberosum L.), and aspen (Populus tremuloides Michx.). However. no information is available on somatic transposition in any plant during several years of growth and development. It is not known how transposition affects genetic variability among vegetative parts that have developed during a long period of growth. In order to explore the possibility of using somatic Ac transposition for gene tagging and mutagenesis in fruit trees, a derivative of the maize Ac transposable element was introduced into 'Duncan' grapefruit (Citrus paradisi Macf.) by Agrobacterium tumefaciens (Smith & Towns.) Conn-mediated stable transformation. Genetically identical 4-year-old sibling trees were established by grafting one of the transformants on Troyer citrange [Citrus sinensis (L.) Osbec. x Poncirus trifoliate (L.) Ras.] rootstocks. We demonstrated that the Ac element was active upon transformation in citrus (Cirrus L.) trees and that transposition can create genetic variability among tree siblings and among leaves collected from different parts of the same tree. Ac was still active among propagated plants 4 years after transformation. clearly indicating that it is capable of maintaining itself in citrus trees for a long period of time. The observation of different integration patterns in different parts of the same tree and within tree siblings originating from the same transformant suggests that an Ac-based mutagenesis system could be very useful in creating somatic mutations in citrus trees.
In the past few years we have analysed alterations in genome structure and expression that occur in wheat upon allopolyploidization. Our major findings in natural and synthetic allopolyploid wheat are reviewed here. It was found that allopolyploidization brings about rapid genome evolution through the instantaneous generation of a variety of cardinal genetic and epigenetic alterations comprising: (1) non-random elimination of coding and non-coding DNA sequences, (2) epigenetic changes such as DNA methylation of coding and non-coding DNA leading, among others, to gene silencing, and (3) activation of retroelements, which in turn alters the expression of adjacent genes. These changes were reproducible, occurring in the F1 hybrids or in the first generation(s) of a series of nascent allopolyploids corresponding to various interspecific and intergeneric combinations. Moreover, these changes were similar to those that occurred twice in nature: first, at the transition from diploid to tetraploid wheat (similar to0.5 Mya) and, second, at the transition from tetraploid to hexaploid wheat (similar to9500 years ago). Elimination of non-coding sequences augments the differentiation of homoeologous chromosomes at the polyploid level, thus increasing the physical divergence between homoeologues and contributing to the diploid-like meiotic behaviour of polyploid wheat. Transcriptional and post-transcriptional alterations of gene activity, including transcriptional activation of retroelements, led to novel expression patterns. These phenomena emphasize the plasticity of the genome with regard to both structure and gene expression. This plasticity in turn might improve the adaptability of the newly formed allopolyploids and facilitate their rapid and successful establishment in nature. (C) 2004 The Linnean Society of London.
Cuticular waxes play a pivotal role in limiting transpirational water loss across the plant surface. The correlation between the chemical composition of the cuticular waxes and their function as a transpiration barrier is still unclear. In the present study, intact tomato fruits (Lycopersicon esculentum) are used, due to their astomatous surface, as a novel integrative approach to investigate this composition-function relationship: wax amounts and compositions of tomato were manipulated before measuring unbiased cuticular transpiration. First, successive mechanical and extractive wax-removal steps allowed the selective modification of epi- and intracuticular wax layers. The epicuticular film consisted exclusively of very-long-chain aliphatics, while the intracuticular compartment contained large quantities of pentacyclic triterpenoids as well. Second, applying reverse genetic techniques, a loss-of-function mutation with a transposon insertion in a very-long-chain fatty acid elongase beta-ketoacyl-CoA synthase was isolated and characterized. Mutant leaf and fruit waxes were deficient in n-alkanes and aldehydes with chain lengths beyond C-30, while shorter chains and branched hydrocarbons were not affected. The mutant fruit wax also showed a significant increase in intracuticular triterpenoids. Removal of the epicuticular wax layer, accounting for one-third of the total wax coverage on wild-type fruits, had only moderate effects on transpiration. By contrast, reduction of the intracuticular aliphatics in the mutant to approximately 50% caused a 4-fold increase in permeability. Hence, the main portion of the transpiration barrier is located in the intracuticular wax layer, largely determined by the aliphatic constituents, but modified by the presence of triterpenoids, whereas epicuticular aliphatics play a minor role.
It is well established that sequence divergence has an inhibitory effect on homologous recombination. However, a detailed analysis of this relationship is missing for most higher eukaryotes. We have measured the rate of somatic recombination between direct repeats as a function of the number, type, and position of divergent nucleotides in Arabidopsis. We show that a minor divergence level of 0.16% (one mutation in otherwise identical 618 bp) has a profound effect, decreasing the recombination rate approximately threefold. A further increase in the divergence level affects the recombination rate to a smaller extent until a "divergence saturation" effect is reached at relatively low levels of divergence (similar to0.5%). The type of mismatched nucleotide does not affect recombination rates. The decrease in the rate of recombination caused by a single mismatch was not affected by the position of the mismatch along the repeat. This suggests that most recombination intermediate tracts contain a mismatch and thus are as long as the full length of the 618-bp repeats. Finally, we could deduce an antirecombination efficiency of similar to66% for the first mismatch in the repeat. Altogether, this work shows some degree of conservation across kingdoms when compared to previous reports in yeast; it also provides new insight into the effect of sequence divergence on homologous recombination.
Two tomato (Lycopersicon esculentum) mutants with dark testae displaying poor germination rate and percentage on both water and 100 mum gibberellin(4+7) were recovered. The mutants were allelic (black seed1-1; bks1-1 and bks1-2), inherited in Mendelian fashion as a recessive gene residing on chromosome 11. They are not allelic to bs (brown seed) -1, -2, or -4, which impair seed germination and possess dark testae. The bks/bs mutants accumulated dark pigment in the cell layers of the testa above the endothelium, which itself accumulated proanthocyanidins similar to wild type. The poor germination performance of bks mutant seeds was because of impediment of the mutant testae to radicle egress. Imbibition on gibberellin(4+7) did not ameliorate germination percentage or rate. The toughening of the bks testa and associated poor germination were partially overcome when seeds were not dried before germination or were dried under N-2 The seeds of the bks mutant have elevated activity of at least one enzyme responsible for the detoxification of reactive oxygen species. The bks mutant is epistatic to 12 anthocyaninless mutants of tomato. Bio- and physicochemical analysis of the bks testa determined that it accumulated a melanic substance. Inheritance of bks/bs mutations contrasts with that of the anthocyaninless mutants, which are inherited according to the genotype of the maternally derived testa. This suggests that the testa manufactures components before its demise that can maximize testa strength, whereas the endosperm/embryo produces factors that are conveyed to the testa, mitigating this process.
Arbuscular mycorrhizae (AM) represent an ancient symbiosis between mycorrhizal fungi and plant roots which co-evolved to exhibit a finely tuned, multistage interaction that assists plant growth. Direct screening efforts for Myc(-) plant mutants resulted in the identification of a tomato (Lycopersicon esculentum L. cv. Micro-Tom) mutant, M20, which was impaired in its ability to support the premycorrhizal infection (pmi) stages. The Myc- phenotype of the M20 mutant was a single Mendelian recessive trait, stable for nine generations, and nonallelic to a previously identified M161 pmi mutant. The M20 mutant was resistant to infection by isolated AM spores and colonized roots. Formation of Glomus intraradices appressoria on M20 roots was normal, as on wild-type (WT) plants, but in significantly reduced numbers. A significant reduction in spore germination was observed in vitro in the presence of M20 exudates relative to WT. Our results indicate that this new mutant shares similar physiological characteristics with the M161 pmi mutant, but has a more suppressive Myc- phenotype response.
Retrotransposons are a principal component of most eukaryotic genomes, representing roughly 40% of the human genome 1 and 50-80% of some grass genomes(2). They are usually transcriptionally silent but can be activated under certain stresses. Despite their considerable contribution to genome structure, their impact on the expression of adjacent genes is not well understood. The steady-state transcript levels originating from Wis 2-1A retrotransposons are much higher in newly synthesized wheat amphiploids (two or more diverged genomes in the same nucleus). On activation, both Wis 2-1A long terminal repeats drive the readout synthesis of new transcripts from adjacent sequences including the antisense or sense strands of known genes. Here we report that activation of these antisense or sense transcripts is associated with silencing or activation of the corresponding genes, respectively. These data, together with the abundance of retrotransposons in genomes and their ability to be activated by various signals, support the view of transposons as potential controlling elements.
Tomato mutants have been used in genetic studies and breeding for decades, yet only a few tomato mutants have been characterized at the molecular level. Similarly, a wealth of sequence information for tomato is now available but the functions of only a few genes are known. New developments - such as the use of saturated mutant populations, new methods for the detection of mutants and new sequence data - are bridging the gap between tomato genes and their functions.
We analyzed the events that affect gene structure and expression in the early stages of allopolyploidy in wheat. The transcriptome response was studies by analyzing 3072 transcripts in the first generation of a synthetic allotetraploid (genome S(1)S(1)A(m)A(n)), which resembles tetraploid wheat (genome BBAA), and in its two diploid progenitors Aegilops sharonensis (S(1)S(1)) and Triticum monococcum ssp. aegilopoides (A(m)A(m)). The expression of 60 out of 3072 transcripts was reproducibly altered in the allotetraploid: 48 transcripts disappeared and 12 were activated. Transcript disappearance was caused by gene silencing or by gene loss. Gene silencing affected one or both homeologous loci and was associated in part with cytosine methylation. Gene loss or methylation had occurred already in the F(1) intergeneric hybrid or in the allotetraploid, depending on the locus. The silenced/lost genes included rRNA genes and genes involved in metabolism, disease resistance, and cell cycle regulation. The activated genes with a known function were all retroelements. These findings show that wide hybridization and chromosome doubling affect gene expression via genetic and epigenetic alterations immediately upon allopolyploid formation. These events contribute to the genetic diploidization of newly formed allopolyploids.
Recent studies have shown that speciation through allopolyploidy, i.e., inter-specific or inter-generic hybridization followed by chromosome doubling, is accompanied by a variety of rapid cardinal genetic and epigenetic changes. This paper reviews our studies on the effect of allopolyploidization on several low-copy, non-coding sequences that exist in all the diploid species of the tribe Triticeae, including the progenitors of polyploid wheat, but in polyploid wheat they occur in only one genome, either in one homologous pair (chromosome-specific sequences) or in several pairs of the same genome (genome-specific sequences). Rapid elimination of these sequences from one genome is a general phenomenon in newly synthesized allopolyploids. Elimination was a nonrandom, reproducible event whose direction was determined by the genomic combination of the amphiploid. It was not affected by the genotype of the parental plants, by their cytoplasm, or by the ploidy level, and it did not result from intergenomic recombination. This elimination augmented the differentiation of homeologous chromosomes (partially homologous chromosomes of the different genomes) at the polyploid level, thus providing the physical basis for the diploid-like meiotic behavior characterizing polyploid wheat. This pattern of pairing prevents intergenomic recombination, and consequently, ensures full fertility, disomic inheritance, and permanent heterosis between alleles of different genomes (homeoalleles). Accordingly, rapid elimination of these sequences improves the fitness of newly formed allopolyploids, facilitating their rapid establishment in nature as new successful species.
Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis. Using 2500 M2 fast-neutron-mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro-Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores. The myc(-) phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus. The mutant exhibited morphological and growth-pattern characteristics similar to those of wild-type plants. Alterations of light intensity and day/night temperatures did not eliminate the myc(-) characteristic. Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita. Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal-inoculated wild-type plants in the same growth medium. During evaluation of the pre-infection stages in the mutant rhizosphere, spore germination and appressoria formation of G. intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild-type plants. These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre-mycorrhizal infection stages.
Selection markers, which were necessary for the isolation of transgenic plants, are no longer required in mature plants, especially when they are grown in fields, Regimes to achieve their efficient elimination. mostly through site-specific recombifiation or transposition, are being developed.
The maize transposon Activator (Ac) was the first mobile DNA element to be discovered. Since then, other elements were found that share similarity to Ac, suggesting that it belongs to a transposon superfamily named hAT after hobo from Drosophila, Ac from maize, and Tam3 from snapdragon. We addressed the structure and evolution of hAT elements by developing new toot for transposon mining and searching the public sequence databases for the hallmarks of hAT elements, namely the transposase and short terminal inverted repeats (TIRs) flanked by 8-bp host duplications. We found 147 MT-related sequences in plants, animals, and fungi. Six conserved blocks could be identified in the transposase of most hAT elements. A total of 41 hAT sequences were flanked by TIRs and 8-bp host duplications and, out of these, 34 sequences had TIRs similar to the consensus determined in this work, suggesting that they are active or recently active transposons. Phylogenetic analysis and clustering of hAT sequences suggest that the hAT superfamily is very ancient, probably predating the plant-fungi-animal separation, and that, unlike previously proposed, there is no evidence that horizontal gene transfer was involved in the evolution of hAT elements.
Interspecific or intergeneric hybridization, followed by chromosome doubling, can lead to the formation of new allopolyploid species. Recent studies indicate that allopolyploid formation is associated with genetic and epigenetic changes, although little is known about the type of changes that occur, how rapidly they occur, and the type of sequences involved. To address these matters, we have surveyed F1 hybrids between diploid species from the wheat (Aegilops and Triticum) group and their derived allotetraploids by screening a large number of loci using amplified fragment length polymorphism and DNA gel blot analysis and by assaying the extent of cytosine methylation. We found that sequence elimination is one of the major and immediate responses of the wheat genome to wide hybridization or allopolyploidy, that it affects a large fraction of the genome, and that it is reproducible. In one cross between Ae. sharonensis x Ae. umbellulata, 14% of the loci from Ae. sharonensis were eliminated compared with only 0.5% from Ae. umbellulata, with most changes occurring in the F1 hybrid. In contrast, crosses between Ae. longissima x T. urartu showed that sequence elimination was more frequent after chromosome doubling. Alterations in cytosine methylation occurred in similar to 13% of the loci, either in the F1 hybrid or in the allopolyploid. For eight of nine bands that were isolated, the sequences that underwent elimination corresponded to low-copy DNA, whereas alterations in methylation patterns affected both repetitive DNA sequences, such as retrotransposons, and low-copy DNA in approximately equal proportions.
To better understand genetic events that accompany allopolyploid formation, we studied the rate and time of elimination of eight DNA sequences in F1 hybrids and newly formed allopolyploids of Aegilops and Triticum. In total, 35 interspecific and intergeneric F1 hybrids and 22 derived allopolyploids were analyzed and compared with their direct parental plants. The studied sequences exist in all the diploid species of the Triticeae but occur in only one genome, either in one homologous pair (chromosome-specific sequences [CSSs]) or in several pairs of the same genome (genome-specific sequences [GSSs]), in the polyploid wheats. It was found that rapid elimination of CSSs and GSSs is a general phenomenon in newly synthesized allopolyploids. Elimination of GSSs was already initiated in F1 plants and was completed in the second or third allopolyploid generation, whereas elimination of CSSs started in the first allopolyploid generation and was completed in the second or third generation. Sequence elimination started earlier in allopolyploids whose genome constitution was analogous to natural polyploids compared with allopolyploids that do not occur in nature. Elimination is a nonrandom and reproducible event whose direction was determined by the genomic combination of the hybrid or the allopolyploid. It was not affected by the genotype of the parental plants, by their cytoplasm, or by the ploidy level, and it did not result from intergenomic recombination. Allopolyploidy-induced sequence elimination occurred in a sizable fraction of the genome and in sequences that were apparently noncoding. This finding suggests a role in augmenting the differentiation of homoeologous chromosomes at the polyploid level, thereby providing the physical basis for the diploid-like meiotic behavior of newly formed allopolyploids. In our view, this rapid genome adjustment may have contributed to the successful establishment of newly formed allopolyploids as new species.
We have isolated a hyperrecombinogenic Nicotiana tabacum mutant. The mutation, Hyrec, is dominant and segregates in a Mendelian fashion. In the mutant, the level of mitotic recombination between homologous chromosomes is increased by more than three orders of magnitude. Recombination between extrachromosomal substrates is increased six- to ninefold, and intrachromosomal recombination is not affected. Hyrec plants were found to perform non-homologous end joining as efficiently as the wild type, ruling out the possibility that the increase in homologous recombination is due to a defect in end joining. In addition, Hyrec plants show significant resistance to gamma-irradiation, whereas UV resistance is not different from the wild type. This suggests that homologous recombination can be strongly up-regulated in plants. Moreover, Hyrec constitutes a novel type of mutation: no similar mutant was reported in plants and hyperrecombinogenic mutants from other organisms usually show sensitivity to DNA damaging agents. We discuss the insight that this mutant provides into understanding the mechanisms of recombination plus the potential application for gene targeting in plants.
The mechanism of transposition of the maize Ac/Ds elements is not well understood. The true transposition intermediates are not known and it has not been possible to distinguish between excision models involving 8-bp staggered cuts or 1-bp staggered cuts followed by hairpin formation, in this work, we have analyzed extrachromosomal excision produces to gain insight into the excision mechanism. Plasmid rescue was used to demonstrate that Ds excision is associated with the formation of circular molecules. Tn addition, we present evidence for the formation of linear extrachromosomal species during Ds excision. Sequences found at the termini of circular and linear elements showed a broad range of nucleotide additions or deletions, suggesting that these species are not true intermediates. Additional nucleotides adjacent to the termini in extrachromosomal elements were compared to the sequence of the original donor site. This analysis showed that: (1) the first nucleotide adjacent to the transposon end was significantly more similar to the first nucleotide nanking the element in the donor site than to a random sequence and (2) the second and farther nucleotides did not resemble the donor site. The implications of these findings for excision models are discussed.
We describe new tools for functional analysis of the tomato genome based on insertional mutagenesis with the maize Ac/Ds transposable elements in the background of the miniature cultivar Micro-Tom. 2932 F-3 families, in which Ds elements transposed and were stabilized, were screened for phenotypic mutations. Out of 10 families that had a clear mutant phenotype, only one mutant was Ds-tagged. In addition, we developed promoter trapping using the firefly luciferase reporter gene and enhancer trapping, using beta-glucuronidase (GUS). We show that luciferase can be used as a non-invasive reporter to identify, isolate and regenerate somatic sectors, to study the time course of mutant expression, and to identify inducible genes. Out of 108 families screened for luciferase activity 55% showed expression in the flower, 11% in the fruit and 4% in seedlings, suggesting a high rate of Ds insertion into genes. Preferential insertion into genes was supported by the analysis of Ds flanking sequences: 28 out of 50 sequenced Ds insertion sites were similar to known genes or to ESTs. In summary, the 2932 lines described here contain 2-3 Ds inserts per line, representing a collection of approximately 7500 Ds insertions. This collection has potential for use in high-throughput functional analysis of genes and promoter isolation in tomato.
Specific binding of plant nuclear proteins to GGTAAA-like motifs in the terminal regions of the transposable elements Ac and Mul has been detected in several laboratories. However, the role of these proteins in transposition remains unknown. To test the hypothesis that this binding activity is necessary for transposition, we identified and mutagenized all the binding motifs within the Dsl element. This analysis enabled us to define more precisely the requirements for binding of the host protein. We then tested the ability of the mutated elements to excise from the maize streak virus (MSV) genome. We found that mutated Dsl elements that do not bind the host proteins, as determined by gel-shift competition assay, are still capable of undergoing excision in maize, although for one of the maize lines the rate of excision was reduced. Excision of mutated Dsl elements generated typical excision footprints. These data indicate that binding of host protein(s) to the GGTAAA-like motifs is not essential for Dsl excision; however, it may contribute to the efficiency of the process.
DNA double-strand breaks (DSBs) lead to serious genomic deficiencies if left unrepaired. Recent studies have provided new insight into the mechanisms, the mutants and the genes involved in DSB repair in plants. These studies indicate that high fidelity DSB repair via homologous recombination is less frequent than non-homologous end-joining. Interestingly, non-homologous end-joining in plants is more error-prone than in other species, being associated with various rearrangements that often include deletions and insertions (filler DNA). We discuss the mechanism of error-prone DSB repair, which is probably an important driving force in plant genome evolution.
Targeted gene disruption exploits homologous recombination (BR) as a powerful reverse genetic tool, for example in bacteria, yeast, and transgenic knockout mice, but it has not been applied to plants, owing to the low frequency of HR and the lack of recombinogenic mutants. To increase the frequency of HR in plants,,ve constructed transgenic tobacco lines carrying the Escherichia coil RuvC gene fused to a plant viral nuclear localization signal. We show that RuvC, encoding an endonuclease that binds to and resolves recombination intermediates (Holiday junctions) is properly transcribed in these lines and stimulates HR We observed a 12-fold stimulation of somatic crossover between genomic sequences, a Ii-fold stimulation of intrachromosomal recombination, and a 56-fold increase for the frequency of extrachromosomal recombination between plasmids cotransformed into young leaves via particle bombardment. This stimulating effect may be transferred to any plant species to obtain recombinogenic plants and thus constitutes an important step toward gene targeting.
The Ac-encoded transposase, a factor that is essential for the mobility of the Ac element, is expressed under the control of a promoter that lacks a conventional TATA box. The regulation of this promoter is poorly understood. We have analyzed Ac promoter structure and activity, both in vitro and in vivo, using transgenic tobacco plants and cell suspensions. A deletion analysis of the Ac 5' region showed that the minimal promoter is located within 70 bp of the major transcription initiation site (at position 334). The minimal promoter includes the sequence TAAGAAATA at position 294-303, i.e., about 30 nucleotides upstream from the transcription start site. This sequence binds specifically to the TATA-binding protein (TBP), suggesting that it is functional as a TATA box. The regulation of the Ac promoter was studied throughout plant development. Levels of Ac mRNA were low in all tissues studied, with higher expression being observed in dividing cells. In order to test whether Ac promoter is regulated during the cell cycle, a tobacco cell suspension transformed with Ac, was grown synchronously. No differences were found in Ac mRNA levels between cells in S, G2, M, or G1 phases; however, expression was lower in the stationary phase. We conclude that Ac promoter is not cell-cycle regulated but is expressed at a higher level in dividing cells. The possible relationship between promoter features and the regulation of Ac element transposition is discussed.
The mechanism by which the maize autonomous Ac transposable element gives rise to nonautonomous Ds elements is largely unknown. Sequence analysis of native maize Ds elements indicates a complex chimeric structure formed through deletions of Ac sequences with or without insertions of Ac-unrelated sequence blocks. These blocks are often flanked by short stretches of reshuffled and duplicated Ac sequences. To better understand the mechanism leading to Ds formation, we designed an assay for detecting alterations in Ac using transgenic tobacco plants carrying a single copy of Ac. We found frequent de novo alterations in Ac which were excision rather than sequence dependent, occurring within Ac but not within an almost identical Ds element and nea, within a stable transposase-producing gene. The de nova DNA rearrangements consisted of internal deletions with breakpoints usually occurring at short repeats and, in some cases, of duplication of Ac sequences or insertion of Ac-unrelated fragments. The ancient maize Ds elements and the young Ds elements in transgenic tobacco showed similar rearrangements, suggesting that Ac-Ds elements evolve rapidly, more so than stable genes, through deletions, duplications, and reshuffling of their own sequences and through capturing of unrelated sequences. The data presented here suggest that abortive Ac-induced gap repair, through the synthesis-dependent strand-annealing pathway, is the underlying mechanism for Ds element formation.
Double strand DNA breaks in plants are primarily repaired via non-homologous end joining, However, little is known about the molecular events underlying this process, We have studied non-homologous end joining of linearized plasmid DNA with different termini configurations following transformation into tobacco cells, A variety of sequences were found at novel end junctions, Joining with no sequence alterations was rare, in most cases, deletions were found at both ends, and rejoining usually occurred at short repeats. A distinct feature of plant junctions was the presence of relatively large, up to 1.2 kb long, insertions (filler DNA), in similar to 30% of the analyzed clones, The filler DNA originated either from internal regions of the plasmid or from tobacco genomic DNA, Some insertions had a complex structure consisting of several reshuffled plasmid-related regions, These data suggest that double strand break repair in plants involves extensive end degradation, DNA synthesis following invasion of ectopic templates and multiple template switches, Such a mechanism is reminiscent of the synthesis-dependent recombination in bacteriophage T4, It can also explain the frequent 'DNA scrambling' associated with illegitimate recombination in plants.
To study genome evolution in allopolyploid plants, we analyzed polyploid wheats and their diploid progenitors for the occurrence of 16 low-copy chromosome- or genome-specific sequences isolated from hexaploid wheat. Based on their occurrence in the diploid species, we classified the sequences into two groups: group I, found in only one of the three diploid progenitors of hexaploid wheat, and group II, found in all three diploid progenitors. The absence of group II sequences from one genome of tetraploid wheat and from two genomes of hexaploid wheat indicates their specific elimination from these genomes at the polyploid level. Analysis of a newly synthesized amphiploid, having a genomic constitution analogous to that of hexaploid wheat, revealed a pattern of sequence elimination similar to the one found in hexaploid wheat. Apparently, speciation through allopolyploidy is accompanied by a rapid, nonrandom elimination of specific, low-copy, probably noncoding DNA sequences at the early stages of allopolyploidization, resulting in further divergence of homoeologous chromosomes (partially homologous chromosomes of different genomes carrying the same order of gene loci). We suggest that such genomic changes may provide the physical basis for the diploid-like meiotic behavior of polyploid wheat.
The prominent repair mechanism of DNA double-strand breaks formed upon excision of the maize Ac transposable element is via nonhomologous end joining. In this work we have studied the role of homologous recombination as an additional repair pathway. To this end, we developed an assay whereby beta-Glucuronidase (GUS) activity is restored upon recombination between two homologous ectopic (nonallelic) sequences in transgenic tobacco plants. One of the recombination partners carried a deletion at the 5' end of GUS and an Ac or a Ds element inserted at the deletion site. The other partner carried an intact 5' end of the GUS open reading frame and had a deletion at the 3' end of the gene. Based on GUS reactivation data, we found that the excision of Ac induced recombination between ectopic sequences by at least tyro orders of magnitude. Recombination events, visualized by blue staining, were detected in seedlings, in pollen and in protoplasts. DNA fragments corresponding to recombination events were recovered exclusively in crosses with Ac-carrying plants, providing physical evidence for Ac-induced ectopic recombination. The occurrence of ectopic recombination following double-strand breaks is a potentially important factor in plant genome evolution.
The maize Ac/Ds transposable elements are thought to transpose via a cut-and-paste mechanism, but the intermediates formed during transposition are still unknown. In this work we present evidence that circular Ac molecules are formed in plants containing actively transposing elements. In these circles, transposon ends are joined head-to-head. The sequence at the ends' junction is variable, containing small deletions or insertions. Circles containing deleted Ac ends are probably unable to successfully reintegrate. To test the ability of circles with intact transposon ends to integrate into tie genome, an artificial Ds circle was constructed by cloning the joined ends of Ac into a plasmid carrying a plant selectable marker. When such a circular Ds was introduced into tobacco protoplasts in the presence of Ac-transposase, no efficient transposase-mediated integration was observed. Although a circular transposition intermediate cannot be ruled out, the findings of circles with deleted transposon ends and the absence of transposase-mediated integration of the circular Ds suggest that some of the joined-ends-carrying elements are not transposition intermediates, but rather abortive excision products. The formation of Ac circles might account for the previously described phenomenon of Ac-loss. The origin of Ac circles and the implications for models of Ac transposition are discussed.
The purpose of this study was to develop a model system for studying tomato genetics. Agronomic, genetic, and molecular data are presented which show that the miniature Lycopersicon esculentum cultivar, Micro-Tom (Micro tomato), fulfills the requirements for such a model. It grows at high density (up to 1357 plants/m(-2)); it has a short life cycle (70-90 days from sowing to fruit ripening); and it can be transformed at frequencies of up to 80% through Agrobacterium-mediated transformation of cotyledons. Moreover, it differs from standard tomato cultivars by only two major genes. Therefore, any mutation or transgene can be conveniently studied in Micro-Tom's background and, when needed, transferred into a standard background. We took advantage of Micro-Tom's features to improve the infrastructure for mutagenesis in tomato. A screening of 9000 M1 and 20 000 M2 EMS mutagenized plants is described. Mutants with altered pigmentation or modified shape of leaves, flowers and fruits were found. In addition, an enhancer trapping and a gene trapping system, based on the Ac/Ds maize transposable elements, were transformed into Micro-Tom and found to be active. In summary, Micro-Tom opens new prospects to achieve saturated mutagenesis in tomato, and facilitates the application of transposon-based technologies such as gene tagging, trapping and knockout.
A mathematical model and a computer simulation were used to study PCR specificity. The model describes the occurrences of non-targeted PCR products formed through random primer-template interactions. The PCR simulation scans DNA sequence databases with primers pairs. According to the model prediction, PCR with complex templates should rarely yield non-targeted products under typical reaction conditions. This is surprising as such products are often amplified in real PCR under conditions optimized for stringency. The causes for this 'PCR paradox' were investigated by comparing the model predictions with simulation results. We found that deviations from randomness in sequences from real genomes could not explain the frequent occurrence of non-targeted products in real PCR. The most likely explanation to the 'PCR paradox' is a relatively high tolerance of PCR to mismatches. The model also predicts that mismatch tolerance has the strongest effect on the number of non-targeted products, followed by primer length, template size and product size limit. The model and the simulation can be utilized for PCR studies, primer design and probing DNA uniqueness and randomness.
Specific binding of Nicotiana nuclear protein(s) to subterminal regions of the Ac transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions of Ac, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding, Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif, The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends of Mutator. Moreover, we show that DNA from Ds1 competes for protein binding to Ac termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region of Tgm1, Tpn1, En/Spm, Tam3 and Ds1-like elements. This suggests that the binding protein(s) might be involved in the transposition process.
Mobility of the maize Ac-Ds transposable element family depends on the production of Ac-encoded transposase (TPASE). The TPASE is a DNA-binding protein which recognizes internal sites near both Ac termini in a region which overlaps the putative TPASE gene promoter. Therefore, it was hypothesized that TPASE may regulate its own transcription. The TPASE effect on Ac promoter activity was tested in transgenic tobacco plants and in protoplasts transformed with Ac-promoter-beta-glucuronidase gene fusions. It was found that TPASE can repress Ac promoter activity in cotyledons and leaves of transgenic plants, as well as in transient assays in protoplasts. TPASE-mediated repression occurs independently of the presence of the Ac untranslated leader or of the 3' termini. When fused to a deleted (-67) cauliflower mosaic virus 35S promoter, the first 237 bp of Ac (starting from the 5' end) are sufficient to enable TPASE-mediated regression. The results indicate that TPASE can act as a transcriptional repressor. The possible mechanisms and significance of TPASE-mediated repression are discussed.
Post-germinative proliferation of cells was studied in cotyledons of Nicotiana tabacum L., Petunia hybrida Vilm. and Arabidopsis thaliana (L.) Heynh. Patterns of cell divisions after germination were characterized by clonal analysis in cotyledons of N. tabacum. The fate of initial cells, which are formed by the end of embryogenesis, was quite variable: cells could undergo between one to seven, and most often, between three to five anticlinal divisions after germination. Sector shape suggested that there were more divisions in length than in width, particularly at the periphery of the cotyledon. The boundaries of clones generated by irradiation of mature seeds did not intersect the midvein, and in most cases, did not intersect lateral veins. The time course of cell divisions during post-germinative development was analyzed cytologically in cotyledons of N. tabacum and P. hybrida. No divisions were detected up to the second day after sowing (DAS), when the radicle emerged. Cotyledon cells started to divide at a rapid rate between 2 and 3 DAS, reaching a mitotic index of about 2% at 3-4 DAS. A rapid decline followed the peak, and no divisions were detected 6-7 DAS. Similarities between leaf and cotyledon development are discussed. In addition, we show that divisions in cotyledons of N. tabacum and A. thaliana chlorophyll mutants can be exploited for a quick and sensitive bioassay from which the effects of various mutagens and DNA repair genes can be assessed.
We report here on the successful painting of a specific plant chromosome within its own genome. Isochromosomes for the long arm of chromosome 5 of the wheat B genome (5BL) were microdissected from first meiotic metaphase spreads of a monoisosomic 5BL line of the common wheat Triticum aestivum cv. Chinese Spring. The dissected isochromosomes were amplified by degenerate oligonucleotide-primed PCR in a single tube reaction. The amplified DNA was used as a complex probe mixture for fluorescent in situ hybridization on first meiotic metaphase spreads of lines carrying 5BL as a distinctive marker. Hybridization signals were observed, specifically, along the entire 5BL. In some of the cells, labeling was also detected in two bivalents, presumably those of the 5B ''homoeologues'' (partial homologues) found in common wheat (5A and 5D). The probe also revealed discrete domains in tapetal nuclei at interphase, further supporting the probe's high specificity. These data suggest that chromosome-and homoeologous group-specific sequences are more abundant in 5BL than genome-specific sequences. Chromosome-painting probes, such as the one described here for 5BL, can facilitate the study of chromosome evolution in polyploid wheat.
Variation in the electrophoretic pattern of the high-molecular-weight (HMW) glutenin subunits was studied in the Ammiad population of wild tetraploid wheat, Triticum turgidum var. dicoccoides (genome AABB), during a 5-year period (1983-4 to 1987-8). These storage proteins were analysed following one-dimensional sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis (SDS PAGE), using seeds collected annually from individual plants at 249 defined sampling sites distributed in 11 habitats. Since plants did mt grow at all the sampling points each year, 1108 accessions were analysed altogether. The population was found to be highly polymorphic: the HMW glutenin loci of genome A, Glu-Al-1 and Glu-Al-2, had four and two alleles. respectively, and those of genome B. Glu-Bl-1 and Glu-Bl-2, had five and seven alleles, respectively. The A-genome alleles appeared in 4 combinations, and the B-genome alleles appeared in 12 combinations. There were 18 intergenomic combinations (A and B genotypes), some of which were very rare while others were abundant and distributed along transects in clusters. The spatial distribution of these genotypes was nonrandom, with each of the 11 habitats characterized by different genotype frequencies. Yearly changes in genotypes, mostly occurring in the last 2 years of the study, had little effect on the total frequencies of die various genotypes. A high affinity was found between specific HMW glutenin genotypes and certain habitats. This affinity may have resulted from a random fixation of specific genotypes in different habitats (founder effect) or, alternatively, from natural selection, thus indicating either linkage between HMW glutenin alleles and adaptive genes (hitchhiking effect) or fitness of some of these allele combinations to specific micro-environments.