High-frequency homologous recombination in plants mediated by zinc-finger nucleases.

Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.

A proteomic analysis of maize chloroplast biogenesis.

Proteomics studies to explore global patterns of protein expression in plant and green algal systems have proliferated within the past few years. Although most of these studies have involved mapping of the proteomes of various organs, tissues, cells, or organelles, comparative proteomics experiments have also led to the identification of proteins that change in abundance in various developmental or physiological contexts. Despite the growing use of proteomics in plant studies, questions of reproducibility have not generally been addressed, nor have quantitative methods been widely used, for example, to identify protein expression classes. In this report, we use the de-etiolation ("greening") of maize (Zea mays) chloroplasts as a model system to explore these questions, and we outline a reproducible protocol to identify changes in the plastid proteome that occur during the greening process using techniques of two-dimensional gel electrophoresis and mass spectrometry. We also evaluate hierarchical and nonhierarchical statistical methods to analyze the patterns of expression of 526 "high-quality," unique spots on the two-dimensional gels. We conclude that Adaptive Resonance Theory 2-a nonhierarchical, neural clustering technique that has not been previously applied to gene expression data-is a powerful technique for discriminating protein expression classes during greening. Our experiments provide a foundation for the use of proteomics in the design of experiments to address fundamental questions in plant physiology and molecular biology.