Exclusion of plastid nucleoids and ribosomes from stromules in tobacco and Arabidopsis.

Stromules are stroma-filled tubules that extend from the surface of plastids and allow the transfer of proteins as large as 550 kDa between interconnected plastids. The aim of the present study was to determine if plastid DNA or plastid ribosomes are able to enter stromules, potentially permitting the transfer of genetic information between plastids. Plastid DNA and ribosomes were marked with green fluorescent protein (GFP) fusions to LacI, the lac repressor, which binds to lacO-related sequences in plastid DNA, and to plastid ribosomal proteins Rpl1 and Rps2, respectively.

Regulation of multiple aquaporin genes in Arabidopsis by a pair of recently duplicated DREB transcription factors.

Identifying the transcription factors that mediate responses to abiotic stress is of fundamental importance in plant biology, not least because of their potential utility in crop improvement. The recently duplicated genes RAP2.4B and RAP2.

Localized hypermutation and associated gene losses in legume chloroplast genomes.

Point mutations result from errors made during DNA replication or repair, so they are usually expected to be homogeneous across all regions of a genome. However, we have found a region of chloroplast DNA in plants related to sweetpea (Lathyrus) whose local point mutation rate is at least 20 times higher than elsewhere in the same molecule. There are very few precedents for such heterogeneity in any genome, and we suspect that the hypermutable region may be subject to an unusual process such as repeated DNA breakage and repair.

High-level expression of Camelid nanobodies in Nicotiana benthamiana.

Nanobodies (or VHHs) are single-domain antigen-binding fragments derived from Camelid heavy chain-only antibodies. Their small size, monomeric behaviour, high stability and solubility, and ability to bind epitopes not accessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. Here we describe high-level expression, in Nicotiana benthamiana, of three versions of an anti-hen egg white lysozyme (HEWL) nanobody which include the original VHH from an immunized library (cAbLys3), a codon-optimized derivative, and a codon-optimized hybrid nanobody comprising the CDRs of cAbLys3 grafted onto an alternative 'universal' nanobody framework.

High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes.

Transgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance. In this article, we explore the potential of transplastomic plants to produce human immunodeficiency virus (HIV) antigens as potential components of an acquired immunodeficiency syndrome (AIDS) vaccine. It is shown that the HIV antigens p24 (the major target of T-cell-mediated immune responses in HIV-positive individuals) and Nef can be expressed to high levels in plastids of tobacco, a non-food crop, and tomato, a food crop with an edible fruit.

The mitochondrial genome of a cytoplasmic male sterile line of perennial ryegrass (Lolium perenne L.) contains an integrated linear plasmid-like element.

The mitochondrial genome of a cytoplasmic male sterile line of perennial ryegrass (Lolium perenne L.) was shown to contain a 9.6 kb element, LpCMSi, that is absent in the mitochondrial genome of fertile lines.

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance.

The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a Delta(9) desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts.

Disruption of essential plastid gene expression caused by T7 RNA polymerase-mediated transcription of plastid transgenes during early seedling development.

Transcription of plastid transgenes by plastid-targeted T7 RNA polymerase (ptT7RNAP) during early seedling development in tobacco was associated with a pale-green leaf phenotype, depletion of plastid rRNAs and arrest of shoot development. Extensive analysis of mutant seedlings at the transcript level using DNA microarrays and RNA gel blotting revealed severe disruption of plastid rRNA accumulation at 4-days post-germination and reduced transcript accumulation for the essential gene clpP. Several nuclear genes encoding plastid proteins were differentially regulated in mutant seedlings over time.

Cocksfoot mottle virus P1 suppresses RNA silencing in Nicotiana benthamiana and Nicotiana tabacum.

The Sobemovirus genome consists of positive sense, single-stranded polycistronic RNA. The 5'-terminal ORF, encoding the protein P1, is its most variable region. Sobemoviral P1 has been described as dispensable for replication but indispensable for systemic infection.

Intron-regulated expression of SUVH3, an Arabidopsis Su(var)3-9 homologue.

SU(VAR)3-9 proteins are key regulators of heterochromatin structure and function in plants, mammals, Drosophila, and yeast. In contrast to animals and fungi, plants contain numerous Su(var)3-9 homologues (SUVH), the members of which form a discrete subfamily. The SU(VAR)3-9 and SUVH proteins associate with heterochromatin and possess histone methyltransferase activity, indicating that they participate in the organization of transcriptionally repressive chromatin.

Gene duplication, exon gain and neofunctionalization of OEP16-related genes in land plants.

OEP16, a channel protein of the outer membrane of chloroplasts, has been implicated in amino acid transport and in the substrate-dependent import of protochlorophyllide oxidoreductase A. Two major clades of OEP16-related sequences were identified in land plants (OEP16-L and OEP16-S), which arose by a gene duplication event predating the divergence of seed plants and bryophytes. Remarkably, in angiosperms, OEP16-S genes evolved by gaining an additional exon that extends an interhelical loop domain in the pore-forming region of the protein.

Transgenic tobacco (Nicotiana tabacum L. cv. Samsun-NN) plants over-expressing a synthetic HRP-C gene are altered in growth, development and susceptibility to abiotic stress.

The physiological role of class III peroxidases (EC 1.11.1.

T7 RNA polymerase-directed expression of an antibody fragment transgene in plastids causes a semi-lethal pale-green seedling phenotype.

A T7 promoter-controlled transgene, AbL, encoding a camel single-domain antibody fragment that binds to the model antigen chicken egg-white lysozyme was introduced into the plastid genome of tobacco. AbL expression was activated in the transplastomic line by introducing a nuclear transgene, ST7, encoding a light-regulated plastid-targeted T7RNAP by cross-pollination. The resulting AbL x ST7 progeny seedlings developed a pale-green phenotype and ceased growth soon after germination.

Mutation of a family 8 glycosyltransferase gene alters cell wall carbohydrate composition and causes a humidity-sensitive semi-sterile dwarf phenotype in Arabidopsis.

The genome of Arabidopsis thaliana contains about 400 genes coding for glycosyltransferases, many of which are predicted to be involved in the synthesis and remodelling of cell wall components. We describe the isolation of a transposon-tagged mutant, parvus, which under low humidity conditions exhibits a severely dwarfed growth phenotype and failure of anther dehiscence resulting in semi-sterility. All aspects of the mutant phenotype were partially rescued by growth under high-humidity conditions, but not by the application of growth hormones or jasmonic acid.

An N-terminal peptide extension results in efficient expression, but not secretion, of a synthetic horseradish peroxidase gene in transgenic tobacco.

Background And Aims: Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.

Evolutionary re-organisation of a large operon in adzuki bean chloroplast DNA caused by inverted repeat movement.

We have sequenced two sections of chloroplast DNA from adzuki bean (Vigna angularis), containing the junctions between the inverted repeat (IR) and large single copy (LSC) regions of the genome. The gene order at both junctions is different from that described for other members of the legume family, such as Lotus japonicus and soybean. These differences have been attributed to an apparent 78-kb inversion that spans nearly the entire LSC region and which is present in adzuki and its close relative, the common bean.

Plastid genes transcribed by the nucleus-encoded plastid RNA polymerase show increased transcript accumulation in transgenic plants expressing a chloroplast-localized phage T7 RNA polymerase.

A gene fusion encoding a plastid-targeted bacteriophage T7 RNA polymerase (T7RNAP) under the transcriptional control of the light-regulated promoter and the plastid-targeting signals of a ribulose-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene was introduced into the nuclear genome of Nicotiana tabacum (tobacco). Immunoblot analysis, in vitro transcription assays and protease treatment of isolated chloroplasts revealed that T7RNAP activity was localized within chloroplasts. RNA gel blot analyses showed a substantial increase in transcript abundance for several plastid genes that are normally transcribed by the nucleus-encoded plastid RNA polymerase (NEP) including rpoC1, rpl33, rps18, rps12, and clpP.