Plant Nuclear Genomes Research Articles

New advances in the production of edible plant vaccines: chloroplast expression of a tetanus vaccine antigen, TetC

Vaccines are a proven method of controlling disease. However there are issues with the delivery and administration of vaccines. A particular problem is that the majority of vaccines currently used are injected, which can be unsafe if needles are reused in areas where blood-borne diseases are prevalent. Vaccines targeting the mucosal immune system avoid many of the problems associated with injections. One potential form of mucosal vaccine is based on the expression of vaccine antigens in plants. Current research in this area has focused on the expression of immunogens from the plant's nuclear genome but low expression levels generally achieved using this system have limited progress. In recent work we have used the model antigen, TetC, which confers resistance to Tetanus infection, to demonstrate the feasibility of expressing vaccine antigens at high levels in the plant chloroplast.

Widespread horizontal transfer of mitochondrial genes in flowering plants.

Horizontal gene transfer--the exchange of genes across mating barriers--is recognized as a major force in bacterial evolution. However, in eukaryotes it is prevalent only in certain phagotrophic protists and limited largely to the ancient acquisition of bacterial genes. Although the human genome was initially reported to contain over 100 genes acquired during vertebrate evolution from bacteria, this claim was immediately and repeatedly rebutted. Moreover, horizontal transfer is unknown within the evolution of animals, plants and fungi except in the special context of mobile genetic elements. Here we show, however, that standard mitochondrial genes, encoding ribosomal and respiratory proteins, are subject to evolutionarily frequent horizontal transfer between distantly related flowering plants. These transfers have created a variety of genomic outcomes, including gene duplication, recapture of genes lost through transfer to the nucleus, and chimaeric, half-monocot, half-dicot genes. These results imply the existence of mechanisms for the delivery of DNA between unrelated plants, indicate that horizontal transfer is also a force in plant nuclear genomes, and are discussed in the contexts of plant molecular phylogeny and genetically modified plants.

An Arabidopsis protein closely related to Synechocystis cryptochrome is targeted to organelles.

Cryptochromes (CRYs) are blue/UV-A photoreceptors related to the DNA repair enzyme DNA photolyase. They have been found in plants, animals and most recently in the cyanobacterium Synechocystis. Closely related to the Synechocystis cryptochrome is the Arabidopsis gene At5g24850. Here, we show that the encoded protein of At5g24850 binds flavin adenine dinucleotide (FAD). It has no photolyase activity, and is likely to function as a photoreceptor. We have named it At-cry3 to distinguish it from the other Arbabidopsis cryptochrome homologues At-cry1 and At-cry2. At-cry3 carries an N-terminal sequence, which mediates import into chloroplasts and mitochondria. Furthermore, we show that At-cry3 binds DNA. DNA binding was also demonstrated for the Synechocystis cryptochrome, indicating that both photoreceptors could have similar modes of action. Based on the finding of a new cryptochrome class in bacteria and plants, it has been suggested that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. However, our phylogenetic analyses are also consistent with an alternative explanation that the presence of cryptochromes in the plant nuclear genome is the result of dual horizontal gene transfer. That is, CRY1 and CRY2 genes may originate from an endosymbiotic ancestor of modern-day alpha-proteobacteria, while the CRY3 gene may originate from an endosymbiotic ancestor of modern-day cyanobacteria.

Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures.

A complementary DNA encoding human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned and introduced into tomato (Lycopersicon esculentum cv. Seokwang) using Agrobacterium-mediated transformation. Genomic PCR and Northern blot analysis demonstrated the integration of the construction into the plant nuclear genome and expression of the hGM-CSF in transgenic tomato. The cell suspension culture was established from leaf-derived calli of the transgenic tomato plants transformed with the hGM-CSF gene. Recombinant hGM-CSF was synthesized by the transgenic cell culture and secreted into the growth medium at 45 microg l(-1) after 10 d' cultivation.

CHLOROPLAST DNA VARIATION IN COCONUT IS OPPOSITE TO ITS NUCLEAR DNA VARIATION

The pattern of world distribution of two major fruit morphotypes of coconuts has led to development of theories on origin, domestication and dissemination of coconut. Results of recent nuclear DNA analyses are in agreement with these theories with several other new insights. Compared to the plant nuclear genome however, the plant organelle genomes, the chloroplast genome and the mitochondrial genome are highly conserved and are maternally inherited in most angiosperms. Therefore, most useful information have come from regions of DNA located in organelle genome for studying phylogeny in angiosperms and for deducing historical information and evolutionary history of populations such as past migration routes and colonization dynamics. This study was aimed to determine the feasibility of developing polymorphic cytoplasmic markers, particularly the chloroplast markers. Chloroplast DNA variation of coconut from all coconut growing regions in the world assessed by both restriction digestions and physical separation of PCR products obtained with universal primers, by chloroplast microsatellites and by sequencing showed no variation. This tends to suggest that coconut may have gone through a severe cytoplasmic bottleneck and only one chloroplast type may have participated in the colonization process.

Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives

Stable integration of a gene into the plant nuclear or chloroplast genome can transform higher plants (e.g. tobacco, potato, tomato, banana) into bioreactors for the production of subunit vaccines for oral or parental administration. This can also be achieved by using recombinant plant viruses as transient expression vectors in infected plants. The use of plant-derived vaccines may overcome some of the major problems encountered with traditional vaccination against infectious diseases, autoimmune diseases and tumours. They also offer a convenient tool against the threat of bio-terrorism. State of the art, experimental strategies, safety and perspectives are discussed in this article.

Genomics, bioinformatics, and plant systematics

Genomics and bioinformatics are emerging disciplines that have the potential to fundamentally alter current approaches to molecular systematics. The sequencing of eukaryotic genomes has demonstrated the ability to obtain and interpret nucleotide sequences at a scale and efficiency that was unimaginable just a few years ago. The immense amount of sequence information generated by genome projects has spurred the development of new analytical and data management tools. How can plant systematists take full advantage of these advances? Comparison of plant nuclear genomes will allow the identification of conserved nonrecombining and single copy loci that can serve as robust phylogenetic markers. The growth of molecular phylogenetic studies is dramatically altering organismal classification. The successful integration of these changes will require innovations in the communication of taxonomic information. Reductions in the cost of obtaining and analyzing nucleotide sequences should result in more time and resources being directed to the essential activities of systematists—the documentation and organization of biological diversity, and the interpretation of this diversity in the context of organismal evolution.

Chloroplast Targeting of Chloroplast Division FtsZ2 Proteins in Arabidopsis

Plant nuclear genomes encode chloroplast division proteins homologous to the eubacterial cell division protein FtsZ. In higher plants, FtsZ genes constitute a small gene family that consists of two subgroups, FtsZ1 and FtsZ2. It was previously hypothesized that members of one family (FtsZ1) targeted chloroplasts, while members of the other family (FtsZ2) localized in the cytoplasm. We determined the full-length cDNA sequences of two FtsZ2 genes from Arabidopsis thaliana (AtFtsZ2-1 and AtFtsZ2-2) and found that the genes encode polypeptides of 478 and 473 amino acids, respectively, and both contain N-terminal extensions beyond what have previously been predicted. The N-terminal regions of both AtFtsZ2-1 and AtFtsZ2-2 were expressed as green fluorescent protein (GFP) fusions under the cauliflower mosaic virus 35S promoter in bombarded tobacco cells. Confocal laser scanning microscopy revealed both fusions exclusively localized to chloroplasts, demonstrating that the N-terminal regions function as chloroplast-targeting signals in vivo. Thus, FtsZ2 proteins function within chloroplasts.

DNA variation in the 5' upstream region of the Adh locus of the wild plants Arabidopsis thaliana and Arabis gemmifera.

To investigate the level and pattern of DNA polymorphism in the noncoding regulatory region in the plant nuclear genome, 2.4 kb of nucleotide sequence of the 5' upstream region of ADH: was determined for 14 ecotypes of Arabidopsis thaliana and five accessions of Arabis gemmifera. Using this data set and previously determined ADH: sequence data, DNA variation was analyzed in a 4.4-kb region of the locus. Two divergent sequence types detected in the transcriptional unit of ADH: were not present in the 5' region of the ADH: gene in A. thaliana. Nucleotide diversity of the entire 5' region was estimated to be 0.0040, which is lower than that in the transcriptional unit. The level of variation was not uniform. There were peaks of variations in a approximately 400-bp region where cis-regulatory elements for ADH: expression were clustered and in exon 4. In interspecific comparison with A. gemmifera, lower divergence was observed in the 5' flanking region than in the exons. High peaks of divergence in the 400-bp regulatory region and exon 4 were also detected, although there were many other peaks. These results indicate that regions of functional importance have a high level of polymorphism and divergence in the ADH: locus of these genera. The possibility of balancing selection in the ADH: gene of these plants is discussed.

Comparative Sequence Analysis of Plant Nuclear Genomes: Microcolinearity and Its Many Exceptions

Comparative Sequence Analysis of Plant Nuclear Genomes: Microcolinearity and Its Many Exceptions

Maize as a model for the evolution of plant nuclear genomes.

The maize genome is replete with chromosomal duplications and repetitive DNA. The duplications resulted from an ancient polyploid event that occurred over 11 million years ago. Based on DNA sequence data, the polyploid event occurred after the divergence between sorghum and maize, and hence the polyploid event explains some of the difference in DNA content between these two species. Genomic rearrangement and diploidization followed the polyploid event. Most of the repetitive DNA in the maize genome is retrotransposable elements, and they comprise 50% of the genome. Retrotransposon multiplication has been relatively recent-within the last 5-6 million years-suggesting that the proliferation of retrotransposons has also contributed to differences in DNA content between sorghum and maize. There are still unanswered questions about repetitive DNA, including the distribution of repetitive DNA throughout the genome, the relative impacts of retrotransposons and chromosomal duplication in plant genome evolution, and the hypothesized correlation of duplication events with transposition. Population genetic processes also affect the evolution of genomes. We discuss how centromeric genes should, in theory, contain less genetic diversity than noncentromeric genes. In addition, studies of diversity in the wild relatives of maize indicate that different genes have different histories and also show that domestication and intensive breeding have had heterogeneous effects on genetic diversity across genes.

A binary-BAC system for plant transformation with high-molecular-weight DNA

A binary-BAC (BIBAC) vector suitable for Agrobacterium-mediated plant transformation with high-molecular-weight DNA was constructed. A BIBAC vector is based on the bacterial artificial chromosome (BAC) library vector and is also a binary vector for Agrobacterium-mediated plant transformation. The BIBAC vector has the minimal origin region of the Escherichia coli F plasmid and the minimal origin of replication of the Agrobacterium rhizogenes Ri plasmid, and thus replicates as a single-copy plasmid in both E. coli and in A. tumefaciens. The T-DNA of the BIBAC vector can be transferred into the plant nuclear genome. As examples, a 30-kb yeast genomic DNA fragment and a 150-kb human genomic DNA fragment were inserted into the BIBAC vector; these constructs were maintained in both E. coli and A. tumefaciens. In order to increase the efficiency of transfer of unusually large BIBAC T-DNAs, helper plasmids that carry additional copies of A. tumefaciens virulence genes virG and virE were constructed. These helper plasmids are compatible with, and can be present in addition to, the BIBAC vector in the A. tumefaciens host. This report details the components of the BIBAC system, providing information essential to the general understanding and the application of this new technology.

Nuclear DNA-based markers for plant evolutionary biology.

While DNA-based markers can provide a wealth of information for the study of plant evolutionary biology, progress is limited by the lack of primers available for PCR. To overcome this limitation, we outline a protocol for developing oligonucleotide primers targeting regions of low copy-number nuclear genes. This protocol is intended to lead to universally useful primer sets. To test our approach, we designed eight primer sets and tested their abilities to amplify targets from representatives of each dicot and one monocot subclass. Five of the eight primer sets amplified targets from at least five of the seven taxa and thus exhibited broad taxonomic usefulness; the remaining primers were rather specific, however, and amplified targets from at most three taxa. In only one primer-taxon combination was a complex multiple-banded amplification produced. Overall, the protocol outlined proved quite useful at identifying broadly applicable primers targeted to low copy-number nuclear genes. Wider application of this approach should be effective at greatly increasing the amount of genetic information available for a diversity of plant nuclear genomes.

Transformation and foreign gene expression in plants by microprojectile bombardment.

AbstractMost common strategies for gene transfer in plants derive from the fact that Agrobacterium tumefaciens contains Ti plasmids which facilitate transfer of T-DNA from the plasmid genome into plant cells with attendant integration of the T-DNA region containing foreign genes into the plant nuclear genome (1). Alternatively, DNA may be introduced into protoplasts using various chemical fusagens (2), by electroporation (3), or by microinjection (4). In all these approaches foreign DNA is introduced into the nuclear genome and mostly single genes driven by independent promoters flanked by polyA-specifying terminators have been expressed. Despite several advantages of the Agrobacterium-mediated transformation, major limitations still remain; the limited host range should be extended to major crop species. Monocots, especially cereals, are highly recalcitrant to Agrobacterium infection. Since regeneration from cultured tissues is a prerequisite, problems of somaclonal variation resulting from tissue culture induced mutations should also be addressed. Direct DNA transfer by electroporation or using chemical fusagens require the use of protoplasts; unfortunately, most crops can not be regenerated from single cells (5).KeywordsChloroplast GenomeForeign GeneImmature EmbryoMultiple Cloning SiteTungsten ParticleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Chloroplast «cryptic» promoter can be activated upon their transfer to plant nuclear genome

Chloroplast «cryptic» promoter can be activated upon their transfer to plant nuclear genome

Stable transfer of intact high molecular weight DNA into plant chromosomes.

In conjunction with an enhanced system for Agrobacterium-mediated plant transformation, a new binary bacterial artificial chromosome (BIBAC) vector has been developed that is capable of transferring at least 150 kb of foreign DNA into a plant nuclear genome. The transferred DNA appears to be intact in the majority of transformed tobacco plants analyzed and is faithfully inherited in the progeny. The ability to introduce high molecular weight DNA into plant chromosomes should accelerate gene identification and genetic engineering of plants and may lead to new approaches in studies of genome organization.

Higher-plant chloroplast and cytosolic 3-phosphoglycerate kinases: a case of endosymbiotic gene replacement

Previous studies indicated that plant nuclear genes for chloroplast and cytosolic isoenzymes of 3-phosphoglycerate kinase (PGK) arose through recombination between a preexisting gene of the eukaryotic host nucleus for the cytosolic enzyme and an endosymbiont-derived gene for the chloroplast enzyme. We readdressed the evolution of eukaryotic pgk genes through isolation and characterisation of a pgk gene from the extreme halophilic, photosynthetic archaebacterium Haloarcula vallismortis and analysis of PGK sequences from the three urkingdoms. A very high calculated net negative charge of 63 for PGK from H. vallismortis was found which is suggested to result from selection for enzyme solubility in this extremely halophilic cytosol. We refute the recombination hypothesis proposed for the origin of plant PGK isoenzymes. The data indicate that the ancestral gene from which contemporary homologues for the Calvin cycle/glycolytic isoenzymes in higher plants derive was acquired by the nucleus from (endosymbiotic) eubacteria. Gene duplication subsequent to separation of Chlamydomonas and land plant lineages gave rise to the contemporary genes for chloroplast and cytosolic PGK isoenzymes in higher plants, and resulted in replacement of the preexisting gene for PGK of the eukaryotic cytosol. Evidence suggesting a eubacterial origin of plant genes for PGK via endosymbiotic gene replacement indicates that plant nuclear genomes are more highly chimaeric, i.e. contain more genes of eubacterial origin, than is generally assumed.

DNA rearrangement associated with the integration of T-DNA in tobacco: an example for multiple duplications of DNA around the integration target.

Transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid is transferred from Agrobacterium tumefaciens to plant cells and is stably integrated into the plant nuclear genome. By the inverse polymerase chain reaction DNA fragments were amplified that contained the T-DNA/plant DNA junctions from the total DNA of a transgenic tobacco plant that had a single copy of the T-DNA in a repetitive region of its genome. A DNA fragment containing the target site was amplified from the total DNA of non-transformed tobacco by the polymerase chain reaction using high-stringency conditions. Comparison of the nucleotide sequence of the target site with those of the T-DNA/plant DNA junctions revealed that various duplications of short stretches of nucleotide sequences around the target and in the incoming T-DNA had accompanied the integration of the T-DNA. A deletion of 16 bp at the target site was also found and the target site was similar, in terms of nucleotide sequence, to regions around the breakpoints of the T-DNA. This finding provides a clear example of the occurrence of complex rearrangements during the integration of T-DNA.

Promiscuous mitochondrial group II intron sequences in plant nuclear genomes.

Gene translocations from the organelles to the nucleus are postulated by the endosymbiont hypothesis. We here report evidence for sequence insertions in the nuclear genomes of plants that are derived from noncoding regions of the mitochondrial genome. Fragments of mitochondrial group II introns are identified in the nuclear genomes of tobacco and a bean species. The duplicated intron sequences of 75-140 bp are derived from cis- and trans-splicing introns of genes encoding subunits 1 and 5 of the NADH dehydrogenase. The mitochondrial sequences are inserted in the vicinities of a lectin gene, different glucanase genes and a gene encoding a subunit of photosystem II. Sequence similarities between the nuclear and mitochondrial copies are in the range of 80 to 97%, suggesting recent transfer events that occurred in the basic glucanase genes before and in the lectin gene after the gene duplications in the evolution of the nuclear gene families. Overlapping regions of the same introns are in two instances also involved in intramitochondrial sequence duplications.

Sequence-tagged site markers for microsatellites: Simplified technique for rapidly obtaining flanking sequences

A sequencing strategy is described for the rapid recovery of DNA sequences flanking repeat sequences of microsatellites in plant nuclear genomes. Primers that represent a perfect microsatellite repeat and end in a 3′ degenerate base have been used to sequence directly from microsatellite repeats in one direction. The procedure allows the design of one flanking primer that is then used to sequence back through the repeat to define the microsatellite site precisely and also provides for the design of the second flanking primer. The strategy is versatile with various repeat sizes and different categories of microsatellites; perfect, imperfect, and compound were found to be suitable templates for analysis.