Summary Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE‐lox site‐specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear‐encoded plastid‐targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA‐ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build‐up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE‐lox system described here will be a highly efficient tool to obtain marker‐free transplastomic plants.

Posttranslational modification is an important element in circadian clock function from cyanobacteria through plants and mammals. For example, a number of key clock components are phosphorylated and thereby marked for subsequent ubiquitination and degradation. Through forward genetic analysis we demonstrate that protein arginine methyltransferase 5 (PRMT5; At4g31120) is a critical determinant of circadian period in Arabidopsis. PRMT5 is coregulated with a set of 1,253 genes that shows alterations in phase of expression in response to entrainment to thermocycles versus photocycles in constant temperature. PRMT5 encodes a type II protein arginine methyltransferase that catalyzes the symmetric dimethylation of arginine residues (Rsme2). Rsme2 modification has been observed in many taxa, and targets include histones, components of the transcription complex, and components of the spliceosome. Neither arginine methylation nor PRMT5 has been implicated previously in circadian clock function, but the period lengthening associated with mutational disruption of prmt5 indicates that Rsme2 is a decoration important for the Arabidopsis clock and possibly for clocks in general.circadian rhythms | circadian clock | luciferase

BackgroundHigh throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination.ResultsWe describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment.ConclusionsExtracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT. We also describe a second bacterial bar gene (b-bar2) and a codon-optimized synthetic bar (s-bar) gene with significantly elevated levels of expression in plastids (Ͼ7% of total soluble cellular protein). Although these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct selection of transgenic clones. The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in codon use. We propose to use codon usage differences as a precautionary measure to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria. Localization of the bar gene in the plastid genome is an attractive alternative to incorporation in the nuclear genome since there is no transmission of plastid-encoded genes via pollen.Bialaphos, a non-selective herbicide, is a tripeptide composed of two l-Ala residues and an analog of Glu known as phosphinothricin (PPT). Bialaphos is toxic to bacteria and plants after intracellular peptidases remove the Ala residues and release active PPT, an inhibitor of Gln synthetase (GS). Inhibition of GS by PPT causes a rapid buildup of intracellular ammonia levels. The associated disruption of chloroplast structure results in inhibition of photosynthesis and plant cell death (Tachibana et al., 1986).Bialaphos-producing species Streptomyces hygroscopicus and Streptomyces viridochromogenes are protected from PPT toxicity by phosphinothricin acetyltransferase (PAT). PAT is encoded by either the bar (bialaphos resistance; Thompson et al., 1987) or pat (phosphinothricin acetyltransferase; Strauch et al., 1988) genes, and detoxifies PPT by acetylation. The PAT enzymes encoded by these two genes are functionally identical and show 85% identity at the amino acid level (Wohlleben et al., 1988; Wehrmann et al., 1996). PPT resistant crops have been obtained by expressing chimeric bar or pat genes in the cytoplasm from nuclear genes. Herbicide resistant lines have been obtained by direct selection for PPT resistance in tobacco (Nicotiana tabacum cv Petit Havana), potato, Brassica napus, Brassica oleracea (De Block et al., 1987; De Block et al., 1989), maize (Spencer et al., 1990), and rice (Cao et al., 1992). Availability of efficient plastid transformation vectors using the spectinomycin resistance (aadA) gene allowed us to test whether or not bar, when expressed in plastids, confers herbicide resistance (S...