MeioSeed: a CellProfiler-based program to count fluorescent seeds for crossover frequency analysis in Arabidopsis thaliana.

BACKGROUND: The formation of crossovers during meiosis is pivotal for the redistribution of traits among the progeny of sexually reproducing organisms. In plants the molecular mechanisms underlying the formation of crossovers have been well established, but relatively little is known about the factors that determine the exact location and the frequency of crossover events in the genome. In the model plant species Arabidopsis, research on these factors has been greatly facilitated by reporter lines containing linked fluorescence marker genes under control of promoters active in seeds or pollen, allowing for the visualization of crossover events by fluorescence microscopy. However, the usefulness of these reporter lines to screen for novel modulators of crossover frequency in a high throughput manner relies on the availability of programs that can accurately count fluorescent seeds. Such a program was previously not available in scientific literature. RESULTS: Here we present MeioSeed, a novel CellProfiler-based program that accurately counts GFP and RFP fluorescent Arabidopsis seeds with adjustable detection thresholds for fluorescence intensity, making use of a robust seed classifier which was trained by machine learning in Ilastik. Using the previously published reporter line Col3-4/20 as an example, we explain the use of MeioSeed and the steps taken to optimize the thresholding settings of the program to fit the published model for recombination frequency and transgene segregation. The use of MeioSeed is illustrated by investigating salt stress as a novel abiotic trigger for changes in crossover frequency in Col3-4/20 (♂) × Ler-0 (♀) F1 hybrids. Salt stress was found to trigger increases in crossover frequency between the marker genes of up to 70% compared to the control treatment without salt stress. Genotyping of control and salt treated populations revealed that the changes in crossover frequency were not limited to the region between the marker genes, but that fluctuations in crossover frequency are likely to occur genome-wide after treatment with high salt concentrations. CONCLUSIONS: MeioSeed allows for the high throughput recognition and counting of fluorescent Arabidopsis seeds and can facilitate the screening for novel abiotic and biotic modulators of crossover frequency using reporter lines in Arabidopsis.

An Arabidopsis mutant with high operating efficiency of Photosystem II and low chlorophyll fluorescence.

The overall light energy to biomass conversion efficiency of plant photosynthesis is generally regarded as low. Forward genetic screens in Arabidopsis have yielded very few mutants with substantially enhanced photochemistry. Here, we report the isolation of a novel Arabidopsis mutant with a high operating efficiency of Photosystem II (φPSII) and low chlorophyll fluorescence from a library of lines harboring T-DNA constructs encoding artificial transcription factors. This mutant was named Low Chlorophyll Fluorescence 1 (LCF1). Only a single T-DNA insertion was detected in LCF1, which interrupted the expression of the full length mRNA of the gene At4g36280 (MORC2). We demonstrate that the high φPSII and low levels of chlorophyll fluorescence were due to a decrease in PSII:PSI ratio. Although LCF1 plants had decreased rosette surface area and biomass under normal growth conditions, they contained more starch per gram fresh weight. The growth defect of LCF1 was alleviated by low light and short day conditions, and growth could even be enhanced after a period of dark-induced senescence, showing that the plant can utilize its excess photosynthetic conversion capacity as a resource when needed.

Enhancement of Arabidopsis growth characteristics using genome interrogation with artificial transcription factors.

The rapidly growing world population has a greatly increasing demand for plant biomass, thus creating a great interest in the development of methods to enhance the growth and biomass accumulation of crop species. In this study, we used zinc finger artificial transcription factor (ZF-ATF)-mediated genome interrogation to manipulate the growth characteristics and biomass of Arabidopsis plants. We describe the construction of two collections of Arabidopsis lines expressing fusions of three zinc fingers (3F) to the transcriptional repressor motif EAR (3F-EAR) or the transcriptional activator VP16 (3F-VP16), and the characterization of their growth characteristics. In total, six different 3F-ATF lines with a consistent increase in rosette surface area (RSA) of up to 55% were isolated. For two lines we demonstrated that 3F-ATF constructs function as dominant in trans acting causative agents for an increase in RSA and biomass, and for five larger plant lines we have investigated 3F-ATF induced transcriptomic changes. Our results indicate that genome interrogation can be used as a powerful tool for the manipulation of plant growth and biomass and that it might supply novel cues for the discovery of genes and pathways involved in these properties.

Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast.

Agrobacterium mediated transformation (AMT) has been embraced by biotechnologists as the technology of choice to introduce or alter genetic traits of plants. However, in plants it is virtually impossible to predetermine the integration site of the transferred T-strand unless one is able to generate a double stranded break (DSB) in the DNA at the site of interest. In this study, we used the model organism Saccharomyces cerevisiae to investigate whether the Agrobacterium mediated translocation of site-specific endonucleases via the type IV secretion system (T4SS), concomitantly with T-DNA transfer is possible and whether this can improve the gene targeting efficiency. In addition to that, the effect of different chromatin states on targeted integration, was investigated. It was found that Agrobacterium mediated translocation of the homing endonuclease I-SceI has a positive effect on the integration of T-DNA via the homologous repair (HR) pathway. Furthermore, we obtained evidence that nucleosome removal has a positive effect on I-SceI facilitated T-DNA integration by HR. Reversely; inducing nucleosome formation at the site of integration removes the positive effect of translocated I-SceI on T-DNA integration.

Involvement of Rad52 in T-DNA circle formation during Agrobacterium tumefaciens-mediated transformation of Saccharomyces cerevisiae.

Agrobacterium tumefaciens cells carrying a tumour inducing plasmid (Ti-plasmid) can transfer a defined region of transfer DNA (T-DNA) to plant cells as well as to yeast. This process of Agrobacterium-mediated transformation (AMT) eventually results in the incorporation of the T-DNA in the genomic DNA of the recipient cells. All available evidence indicates that T-strand transfer closely resembles conjugal DNA transfer as found between Gram-negative bacteria. However, where conjugal plasmid DNA transfer starts via relaxase-mediated processing of a single origin of transfer (oriT), the T-DNA is flanked by two imperfect direct border repeats which are both substrates for the Ti-plasmid encoded relaxase VirD2. Yeast was used as a model system to investigate the requirements of the recipient cell for the formation of T-DNA circles after AMT. It was found that, despite the absence of self-homology on the T-DNA, the homologous repair proteins Rad52 and Rad51 are involved in T-DNA circle formation. A model is presented involving the formation of T-DNA concatemers derived from T-strands by a process of strand-transfer catalysed by VirD2. These concatemers are then resolved into T-DNA circles by homologous recombination in the recipient cell.