John C. Gray

Many Parallel Losses of infA from Chloroplast DNA during Angiosperm Evolution with Multiple Independent Transfers to the Nucleus

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA, which codes for translation initiation factor 1, in >300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in ∼24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants.

Structure and topology of cytochrome f in pea chloroplast membranes

A transmembrane arrangement of cytochrome f in chloroplast thylakoid membranes, with the N-terminal heme-containing region in the intrathylakoid space and a 15 amino acid C-terminal sequence in the stroma, is suggested by the amino acid sequence deduced from the nucleotide sequence of the pea chloroplast gene. This topology has been confirmed by partial proteolysis of the polypeptide in intact and disrupted thylakoid membranes and in inside-out and right-side-out vesicles of chloroplast membranes.

Control of Isoprenoid Biosynthesis in Higher Plants

Publisher Summary Higher plants contain a bewildering array of isoprenoid compounds with a wide variety of structures and functions. In higher plants, many of these isoprenoid compounds play vital roles in the metabolism and development of the plant. The plant growth regulators, abscisic acid and gibberellins, are isoprenoid compounds and many cytokinins contain an isoprenoid side chain. Isoprenoid side chains are also found in many other biologically active molecules; including chlorophylls, plastoquinone, and other prenylquinones in chloroplasts, where together with carotenoids they are involved in photosynthesis. Isoprenoid side chains are also found in the mitochondria1 electron transfer chain components, ubiquinone, and haem α of cytochrome oxidase. Plants must be able to produce this wide range of isoprenoid compounds in different amounts in different parts of the plant at different stages of growth and development. As all these compounds are produced by a common biosynthetic pathway, the plant must have exquisite control mechanisms to ensure the synthesis of the necessary compounds in the right place at the right time. This chapter considers the knowledge of the control of isoprenoid biosynthesis in higher plants, particularly with respect to the properties and subcellular locations of the enzymes involved.

GRASSIUS: A Platform for Comparative Regulatory Genomics across the Grasses

Transcription factors (TFs) are major players in gene regulatory networks and interactions between TFs and their target genes furnish spatiotemporal patterns of gene expression. Establishing the architecture of regulatory networks requires gathering information on TFs, their targets in the genome, and the corresponding binding sites. We have developed GRASSIUS (Grass Regulatory Information Services) as a knowledge-based Web resource that integrates information on TFs and gene promoters across the grasses. In its initial implementation, GRASSIUS consists of two separate, yet linked, databases. GrassTFDB holds information on TFs from maize (Zea mays), sorghum (Sorghum bicolor), sugarcane (Saccharum spp.), and rice (Oryza sativa). TFs are classified into families and phylogenetic relationships begin to uncover orthologous relationships among the participating species. This database also provides a centralized clearinghouse for TF synonyms in the grasses. GrassTFDB is linked to the grass TFome collection, which provides clones in recombination-based vectors corresponding to full-length open reading frames for a growing number of grass TFs. GrassPROMDB contains promoter and cis-regulatory element information for those grass species and genes for which enough data are available. The integration of GrassTFDB and GrassPROMDB will be accomplished through GrassRegNet as a first step in representing the architecture of grass regulatory networks. GRASSIUS can be accessed from www.grassius.org.

A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation

BackgroundThe floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7–10 d and high seedling density and fungal contamination may result in failure to recover transformants.ResultsA method for identifying transformed seedlings in as little as 3.25 d has been developed. Arabidopsis T1 seeds obtained after floral dip transformation are plated on 1% agar containing MS medium and kanamycin, phosphinothricin or hygromycin B, as appropriate. After a 2-d stratification period, seeds are subjected to a regime of 4–6 h light, 48 h dark and 24 h light (3.25 d). Kanamycin-resistant and phosphinothricin-resistant seedlings are easily distinguished from non-resistant seedlings by green expanded cotyledons whereas non-resistant seedlings have pale unexpanded cotyledons. Seedlings grown on hygromycin B differ from those grown on kanamycin and phosphinothricin as both resistant and non-resistant seedlings are green. However, hygromycin B-resistant seedlings are easily identified as they have long hypocotyls (0.8–1.0 cm) whereas non-resistant seedlings have short hypocotyls (0.2–0.4 cm).ConclusionThe method presented here is an improvement on current selection methods as it allows quicker identification of transformed seedlings: transformed seedlings are easily discernable from non-transformants in as little as 3.25 d in comparison to the 7–10 d required for selection using current protocols.

The ancestral symbiont sensor kinase CSK links photosynthesis with gene expression in chloroplasts.

We describe a novel, typically prokaryotic, sensor kinase in chloroplasts of green plants. The gene for this chloroplast sensor kinase (CSK) is found in cyanobacteria, prokaryotes from which chloroplasts evolved. The CSK gene has moved, during evolution, from the ancestral chloroplast to the nuclear genomes of eukaryotic algae and green plants. The CSK protein is now synthesised in the cytosol of photosynthetic eukaryotes and imported into their chloroplasts as a protein precursor. In the model higher plant Arabidopsis thaliana, CSK is autophosphorylated and required for control of transcription of chloroplast genes by the redox state of an electron carrier connecting photosystems I and II. CSK therefore provides a redox regulatory mechanism that couples photosynthesis to gene expression. This mechanism is inherited directly from the cyanobacterial ancestor of chloroplasts, is intrinsic to chloroplasts, and is targeted to chloroplast genes.

Stable Plastid Transformation in Lettuce (Lactuca sativa L.)

Although plastid transformation in higher plants was first demonstrated in the early 1990s it is only recently that the technology is being extended to a broader range of species. To date, the production of fertile transplastomic plants has been reported for tobacco, tomato, petunia, soybean, cotton and Lesquerella fendleri (Brassicaceae). In this study we demonstrate a polyethylene glycol-mediated plastid transformation system for lettuce that generates fertile, homoplasmic, plastid-transformed lines. Transformation was achieved using a vector that targets genes to the trnA/trnI intergenic region of the lettuce plastid genome employing the aadA gene as a selectable marker against spectinomycin. Spectinomycin resistance and heterologous gene transcription were shown in T1 plants derived from self-pollinated primary regenerants demonstrating transmission of the plastid-encoded transgene to the first seed generation. Crossing with male sterile wild-type lettuce showed that spectinomycin resistance was not transmitted via pollen. Constructs containing the gfp gene showed plastid-based expression of green fluorescent protein. The lettuce plastid could have potential both as a production and a delivery system for edible human therapeutic proteins.

Plastid Translation Is Required for the Expression of Nuclear Photosynthesis Genes in the Dark and in Roots of the Pea lip1 Mutant

The expression of nuclear photosynthesis genes in pea seedlings requires both light and a postulated signal produced by developing plastids. The requirement for the plastid signal for the accumulation of transcripts of Lhcb1, RbcS, PetE, and AtpC genes was investigated in the pea mutant lip1, which shows light-independent photomorphogenic development. Lincomycin and erythromycin, inhibitors of plastid translation, decreased the accumulation of transcripts of nuclear photosynthesis genes in shoots of light-grown wild-type and lip1 seedlings, indicating that the plastid signal is required in the lip1 mutant. Treatment with lincomycin or erythromycin also reduced the accumulation of transcripts in shoots of dark-grown lip1 seedlings, indicating that light is not an obligate requirement for the synthesis or activity of the plastid signal. Lincomycin had a similar effect on the accumulation of Lhcb1 transcripts in dark-grown cop1-4 seedlings of Arabidopsis. Accumulation of transcripts of nuclear photosynthesis genes was also observed in roots of light-grown lip1 seedlings, and this accumulation, which was associated with the development of chloroplasts, was again dependent on plastid translation. The plastid signal therefore regulates the expression of nuclear photosynthesis genes in the dark and in roots of the lip1 mutant.

A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes.

A galinstan expansion femtosyringe enables femtoliter to attoliter samples to be introduced into prokaryotes and subcellular compartments of eukaryotes. The method uses heat-induced expansion of galinstan (a liquid metal alloy of gallium, indium, and tin) within a glass syringe to expel samples through a tip diameter of about 0.1 μm. The narrow tip inflicts less damage than conventional capillaries, and the heat-induced expansion of the galinstan allows fine control over the rate of injection. We demonstrate injection of Lucifer Yellow and Lucifer Yellow–dextran conjugates into cyanobacteria, and into nuclei and chloroplasts of higher organisms. Injection of a plasmid containing the bla gene into the cyanobacterium Phormidium laminosum resulted in transformed ampicillin-resistant cultures. Green fluorescent protein was expressed in attached leaves of tobacco and Vicia faba following injection of DNA cantaining its gene into individual chloroplasts.

Sequence of the Tomato Chloroplast DNA and Evolutionary Comparison of Solanaceous Plastid Genomes

Tomato, Solanum lycopersicum (formerly Lycopersicon esculentum), has long been one of the classical model species of plant genetics. More recently, solanaceous species have become a model of evolutionary genomics, with several EST projects and a tomato genome project having been initiated. As a first contribution toward deciphering the genetic information of tomato, we present here the complete sequence of the tomato chloroplast genome (plastome). The size of this circular genome is 155,461 base pairs (bp), with an average AT content of 62.14%. It contains 114 genes and conserved open reading frames (ycfs). Comparison with the previously sequenced plastid DNAs of Nicotiana tabacum and Atropa belladonna reveals patterns of plastid genome evolution in the Solanaceae family and identifies varying degrees of conservation of individual plastid genes. In addition, we discovered several new sites of RNA editing by cytidine-to-uridine conversion. A detailed comparison of editing patterns in the three solanaceous species highlights the dynamics of RNA editing site evolution in chloroplasts. To assess the level of intraspecific plastome variation in tomato, the plastome of a second tomato cultivar was sequenced. Comparison of the two genotypes (IPA-6, bred in South America, and Ailsa Craig, bred in Europe) revealed no nucleotide differences, suggesting that the plastomes of modern tomato cultivars display very little, if any, sequence variation.