The role of AtMUS81 in interference-insensitive crossovers in A. thaliana.

MUS81 is conserved among plants, animals, and fungi and is known to be involved in mitotic DNA damage repair and meiotic recombination. Here we present a functional characterization of the Arabidopsis thaliana homolog AtMUS81, which has a role in both mitotic and meiotic cells. The AtMUS81 transcript is produced in all tissues, but is elevated greater than 9-fold in the anthers and its levels are increased in response to gamma radiation and methyl methanesulfonate treatment. An Atmus81 transfer-DNA insertion mutant shows increased sensitivity to a wide range of DNA-damaging agents, confirming its role in mitotically proliferating cells. To examine its role in meiosis, we employed a pollen tetrad-based visual assay. Data from genetic intervals on Chromosomes 1 and 3 show that Atmus81 mutants have a moderate decrease in meiotic recombination. Importantly, measurements of recombination in a pair of adjacent intervals on Chromosome 5 demonstrate that the remaining crossovers in Atmus81 are interference sensitive, and that interference levels in the Atmus81 mutant are significantly greater than those in wild type. These data are consistent with the hypothesis that AtMUS81 is involved in a secondary subset of meiotic crossovers that are interference insensitive.

Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis.

Recombination, in the form of cross-overs (COs) and gene conversion (GC), is a highly conserved feature of meiosis from fungi to mammals. Recombination helps ensure chromosome segregation and promotes allelic diversity. Lesions in the recombination machinery are often catastrophic for meiosis, resulting in sterility. We have developed a visual assay capable of detecting Cos and GCs and measuring CO interference in Arabidopsis thaliana. This flexible assay utilizes transgene constructs encoding pollen-expressed fluorescent proteins of three different colors in the qrt1 mutant background. By observing the segregation of the fluorescent alleles in 92,489 pollen tetrads, we demonstrate (i) a correlation between developmental position and CO frequency, (ii) a temperature dependence for CO frequency, (iii) the ability to detect meiotic GC events, and (iv) the ability to rapidly assess CO interference.

Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene.

Arabidopsis (Arabidopsis thaliana) QUARTET (QRT) genes are required for pollen separation during normal floral development. In qrt mutants, the four products of microsporogenesis remain fused and pollen grains are released as tetrads. In Arabidopsis, tetrad analysis in qrt mutants has been used to map all five centromeres, easily distinguish sporophytic from gametophytic mutations, and accurately assess crossover interference. Using a combination of forward and reverse genetics, we have identified the gene responsible for the qrt1 phenotype. Annotation predicts that QRT1 encodes a pectin methylesterase (PME), and enzymatic assays of QRT1 expressed in Escherichia coli indicate that QRT1 has PME activity. Promoter and transcription analysis demonstrate QRT1 is expressed in anther tissues shortly after meiosis is complete. Unexpectedly, the QRT1 promoter is also active in a variety of developmentally unrelated tissues, including developing guard cells, the hypocotyl-root transition zone, areas of lateral root emergence, and floral nectaries. PMEs constitute a large gene family in Arabidopsis, are involved in cell wall loosening, and have been implicated in various aspects of floral development and pollen tube elongation. The identification of QRT1 as a PME contributes to our understanding of pollen development and may help to provide valuable genetic tools in other plant species.

Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T-DNA integrations.

Several recent investigations of T-DNA integration sites in Arabidopsis thaliana have reported 'cold spots' of integration, especially near centromeric regions. These observations have contributed to the ongoing debate over whether T-DNA integration is random or occurs preferentially in transcriptionally active regions. When transgenic plants are identified by selecting or screening for transgenic activity, transformants with integrations into genomic regions that suppress transcription, such as heterochromatin, may not be identified. This phenomenon, which we call selection bias, may explain the perceived non-random distribution of T-DNA integration in previous studies. In order to investigate this possibility, we have characterized the sites of T-DNA integration in the genomes of transgenic plants identified by pooled polymerase chain reaction (PCR), a procedure that does not require expression of the transgene, and is therefore free of selection bias. Over 100 transgenic Arabidopsis plants were identified by PCR and compared with kanamycin-selected transformants from the same T(1) seed pool. A higher perceived transformation efficiency and a higher frequency of transgene silencing were observed in the PCR-identified lines. Together, the data suggest approximately 30% of transformation events may result in non-expressing transgenes that would preclude identification by selection. Genomic integration sites in PCR-identified lines were compared with those in existing T-DNA integration databases. In PCR-identified lines with silenced transgenes, the integration sites mapped to regions significantly underrepresented by T-DNA integrations in studies where transformants were identified by selection. The data presented here suggest that selection bias can account for at least some of the observed non-random integration of T-DNA into the Arabidopsis genome.