Complete plastome sequences of Equisetum arvense and Isoetes flaccida: implications for phylogeny and plastid genome evolution of early land plant lineages.

BACKGROUND: Despite considerable progress in our understanding of land plant phylogeny, several nodes in the green tree of life remain poorly resolved. Furthermore, the bulk of currently available data come from only a subset of major land plant clades. Here we examine early land plant evolution using complete plastome sequences including two previously unexamined and phylogenetically critical lineages. To better understand the evolution of land plants and their plastomes, we examined aligned nucleotide sequences, indels, gene and nucleotide composition, inversions, and gene order at the boundaries of the inverted repeats. RESULTS: We present the plastome sequences of Equisetum arvense, a horsetail, and of Isoetes flaccida, a heterosporous lycophyte. Phylogenetic analysis of aligned nucleotides from 49 plastome genes from 43 taxa supported monophyly for the following clades: embryophytes (land plants), lycophytes, monilophytes (leptosporangiate ferns + Angiopteris evecta + Psilotum nudum + Equisetum arvense), and seed plants. Resolution among the four monilophyte lineages remained moderate, although nucleotide analyses suggested that P. nudum and E. arvense form a clade sister to A. evecta + leptosporangiate ferns. Results from phylogenetic analyses of nucleotides were consistent with the distribution of plastome gene rearrangements and with analysis of sequence gaps resulting from insertions and deletions (indels). We found one new indel and an inversion of a block of genes that unites the monilophytes. CONCLUSIONS: Monophyly of monilophytes has been disputed on the basis of morphological and fossil evidence. In the context of a broad sampling of land plant data we find several new pieces of evidence for monilophyte monophyly. Results from this study demonstrate resolution among the four monilophytes lineages, albeit with moderate support; we posit a clade consisting of Equisetaceae and Psilotaceae that is sister to the "true ferns," including Marattiaceae.

Comparison of gene order of GIGANTEA loci in yellow-poplar, monocots, and eudicots.

GIGANTEA plays an important role in the control of circadian rhythms and photoperiodic flowering. The GIGANTEA gene has been studied in various species, but not in basal angiosperms. Moreover, to the best of our knowledge, no study of the genome organization of a basal angiosperm has yet been published. In this study, we sequenced a bacterial artificial chromosome (BAC) harboring GIGANTEA from yellow-poplar (Liriodendron tulipifera L.) and compared the genomic organization of this gene in yellow-poplar with that in other species from various angiosperm clades. This is the first report on the gene structure and organization of a large contig in any basal angiosperm species. The BAC clone, covering a region of approximately 122 kb from the yellow-poplar genome, was sequenced and assembled by coupling the 454 pyrosequencing technology with ABI capillary sequencing. In addition to GIGANTEA, the gene RPS18.A (encoding ribosomal protein S18.A) was found in this segment of the genome. We found that gene content and order in this region of the yellow-poplar genome were similar to those in the corresponding region in eudicots but not in Oryza sativa and Sorghum bicolor, implying that clustering of the GIGANTEA and RPS18.A genes is ancestral and separation of the genes occurred after the phylogenetic split of monocots from dicots. Phylogenetic analysis of GIGANTEA amino acid sequences placed yellow-poplar closer to eudicots than to monocots. In addition, evidence for transposition and large insertions and duplications was found, suggesting multiple and complex mechanisms of basal angiosperm genome evolution.

Chloroplast genome sequence of the moss Tortula ruralis: gene content, polymorphism, and structural arrangement relative to other green plant chloroplast genomes.

BACKGROUND: Tortula ruralis, a widely distributed species in the moss family Pottiaceae, is increasingly used as a model organism for the study of desiccation tolerance and mechanisms of cellular repair. In this paper, we present the chloroplast genome sequence of T. ruralis, only the second published chloroplast genome for a moss, and the first for a vegetatively desiccation-tolerant plant. RESULTS: The Tortula chloroplast genome is approximately 123,500 bp, and differs in a number of ways from that of Physcomitrella patens, the first published moss chloroplast genome. For example, Tortula lacks the approximately 71 kb inversion found in the large single copy region of the Physcomitrella genome and other members of the Funariales. Also, the Tortula chloroplast genome lacks petN, a gene found in all known land plant plastid genomes. In addition, an unusual case of nucleotide polymorphism was discovered. CONCLUSIONS: Although the chloroplast genome of Tortula ruralis differs from that of the only other sequenced moss, Physcomitrella patens, we have yet to determine the biological significance of the differences. The polymorphisms we have uncovered in the sequencing of the genome offer a rare possibility (for mosses) of the generation of DNA markers for fine-level phylogenetic studies, or to investigate individual variation within populations.

Spectroscopic and biochemical analysis of regions of the cell wall of the unicellular 'mannan weed', Acetabularia acetabulum.

Although the Dasycladalean alga Acetabularia acetabulum has long been known to contain mannan-rich walls, it is not known to what extent wall composition varies as a function of the elaborate cellular differentiation of this cell, nor has it been determined what other polysaccharides accompany the mannans. Cell walls were prepared from rhizoids, stalks, hairs, hair scars, apical septa, gametophores and gametangia, subjected to nuclear magnetic resonance and Fourier transform infrared spectroscopy, and analyzed for monosaccharide composition and linkage, although material limitations prevented some cell regions from being analyzed by some of the methods. In diplophase, walls contain a para-crystalline mannan, with other polysaccharides accounting for 10-20% of the wall mass; in haplophase, gametangia have a cellulosic wall, with mannans and other polymers representing about a quarter of the mass. In the walls of the diplophase, the mannan appears less crystalline than typical of cellulose. The walls of both diploid and haploid phases contain little if any xyloglucan or pectic polysaccharides, but appear to contain small amounts of a homorhamnan, galactomannans and glucogalactomannans, and branched xylans. These ancillary polysaccharides are approximately as abundant in the cellulose-rich gametangia as in the mannan-rich diplophase. In the diplophase, different regions of the cell differ modestly but reproducibly in the composition of the cell wall. These results suggest unique cell wall architecture for the mannan-rich cell walls of the Dasycladales.

The first complete chloroplast genome sequence of a lycophyte, Huperzia lucidula (Lycopodiaceae).

We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8x depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373 bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,657 bp. Gene order is more similar to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperzia chloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophyte chloroplast genome data also enable a better reconstruction of the basal tracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, inferred amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.

Development and organization of the central vacuole of Acetabularia acetabulum.

* Here we analyzed the shape of the central vacuole of Acetabularia acetabulum by visualizing its development during diplophase (from juvenility through reproduction) and haplophase (from meiosis through mating). * Light microscopy and whole-organism applications of a pH-sensitive dye, neutral red, were used to visualize the anatomy of the central vacuole. We studied connectivity within the thallus by locally applying dye to morphologically distinct regions (rhizoid, stalk, apex, hairs) and observing dye movements. * In vegetative thalli most of the rhizoid, stalk and young hairs stained with dye. In reproductive structures (caps, gametangia) dye also stained the majority of the interiors. When applied to small areas, dye moved at different rates through each region of the thallus (e.g. within the stalk). Dye moved from younger hairs, but not from older hairs, into the stalk. Errors in incorporation of central vacuole into gametangia occurred at <10(-5). * These data indicate that the central vacuole of A. acetabulum is a ramified polar organelle with, potentially, a gel-like sap that actively remodels its morphology during development.

X-ray absorption near-edge structure analysis of arsenic species for application to biological environmental samples.

Arsenic is an element that is ubiquitous in the environment and is known to form compounds with toxic, even carcinogenic properties. Arsenic toxicity is a function of its chemical form (species). Identification of arsenic species is necessary to accurately determine the transformation and fate of arsenicals as well as the actual risk posed by arsenic contamination. We report X-ray absorption near-edge structure (XANES) measurements of 16 biologically important arsenic compounds. Solid and aqueous standards were studied for differences in XANES spectral features, white line positions, stability during exposure to the beam, and stability between two beam exposures separated by 48 h. Samples containing As(III) (11870.0-11871.7+/-0.5 eV) and As(V) (11872.6-11875.3+/-0.5 eV) were easily distinguished by white line energies and could be further subdivided into a total of seven groups. Valuable examples include As(III)-sulfur compounds (11870.0+/-0.5 eV), arsenobetaine and arsenocholine (11872.6+/-0.5 eV), and a dimethyl arsinyl riboside (11873.3+/-0.5 eV). A growing number of environmental and biological studies use X-ray absorption spectroscopy (XAS) results to complement their more traditional analyses. Results provided here are intended to help make XAS more accessible to new users interested in the study of arsenic in the environment.

Comparison of ESTs from juvenile and adult phases of the giant unicellular green alga Acetabularia acetabulum.

BACKGROUND: Acetabularia acetabulum is a giant unicellular green alga whose size and complex life cycle make it an attractive model for understanding morphogenesis and subcellular compartmentalization. The life cycle of this marine unicell is composed of several developmental phases. Juvenile and adult phases are temporally sequential but physiologically and morphologically distinct. To identify genes specific to juvenile and adult phases, we created two subtracted cDNA libraries, one adult-specific and one juvenile-specific, and analyzed 941 randomly chosen ESTs from them. RESULTS: Clustering analysis suggests virtually no overlap between the two libraries. Preliminary expression data also suggests that we were successful at isolating transcripts differentially expressed between the two developmental phases and that many transcripts are specific to one phase or the other. Comparison of our EST sequences against publicly available sequence databases indicates that ESTs from the adult and the juvenile libraries partition into different functional classes. Three conserved sequence elements were common to several of the ESTs and were also found within the genomic sequence of the carbonic anhydrase1 gene from A. acetabulum. To date, these conserved elements are specific to A. acetabulum. CONCLUSIONS: Our data provide strong evidence that adult and juvenile phases in A. acetabulum vary significantly in gene expression. We discuss their possible roles in cell growth and morphogenesis as well as in phase change. We also discuss the potential role of the conserved elements found within the EST sequences in post-transcriptional regulation, particularly mRNA localization and/or stability.

Precious cells contain precious information: strategies and pitfalls in expression analysis from a few cells.

Expression analysis, often encompassed in the term "functional genomics," is the link between physiology and molecular biology. Often, specific physiological changes in plant development are due to a limited number of genes, expressed exclusively in very few cells of an organ or organism. Compounding the situation, these physiological changes may also be transient. Therefore, searching for the responsible genes, though exciting and necessary to understand important processes, is hindered primarily by the scarcity of "precious" cells in the desired physiological state. Used judiciously, molecular methods such as reverse transcription polymerase chain reaction (RT-PCR), microarray analysis, or subtractive hybridization allow analysis of rare or special cells. Each of these methods has its advantages and pitfalls. Working with precious cells entails special biological strategies to avoid excessive work in obtaining the data and misinterpretation of it. To illustrate the logic and methods involved in working with precious cells-tissues, we describe how subtractive hybridization followed by expressed sequence tag (EST) sequencing can be used to search for a few genes specific to a few available cells.

ELABORATION OF BODY PLAN AND PHASE CHANGE DURING DEVELOPMENT OF ACETABULARIA: How Is the Complex Architecture of a Giant Unicell Built?

While uninucleate and unicellular, Acetabularia acetabulum establishes and maintains functionally and morphologically distinct body regions and executes phase changes like those in vascular plants. Centimeters tall at maturity, this species has allowed unusual experimental approaches. Amputations revealed fates of nucleate and enucleate portions from both wild type and mutants. Historically, graft chimeras between nucleate and enucleate portions suggested that morphological instructions were supplied by the nucleus but resided in the cytoplasm and could be expressed interspecifically. Recently, graft chimeras enabled rescue of mutants arrested in vegetative phase. Since the 1930s, when Acetabularia provided the first evidence for the existence of mRNAs, a dogma has arisen that it uses long-lived mRNAs to effect morphogenesis. While the evidence favors translational control, the postulated mRNAs have not been identified, and the mechanism of morphogenesis remains unknown. Amenable to biochemistry, physiology, and both classical and molecular genetics, Acetabularia may contribute yet new insights into plant development and morphogenesis.