Dina F. Mandoli

Construction of a bacterial artificial chromosome library from the spikemoss Selaginella moellendorffii: a new resource for plant comparative genomics

BackgroundThe lycophytes are an ancient lineage of vascular plants that diverged from the seed plant lineage about 400 Myr ago. Although the lycophytes occupy an important phylogenetic position for understanding the evolution of plants and their genomes, no genomic resources exist for this group of plants.ResultsHere we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from the lycophyte Selaginella moellendorffii. Based on cell flow cytometry, this species has the smallest genome size among the different lycophytes tested, including Huperzia lucidula, Diphaiastrum digita, Isoetes engelmanii and S. kraussiana. The arrayed BAC library consists of 9126 clones; the average insert size is estimated to be 122 kb. Inserts of chloroplast origin account for 2.3% of the clones. The BAC library contains an estimated ten genome-equivalents based on DNA hybridizations using five single-copy and two duplicated S. moellendorffii genes as probes.ConclusionThe S. moellenforffii BAC library, the first to be constructed from a lycophyte, will be useful to the scientific community as a resource for comparative plant genomics and evolution.

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

BackgroundTortula 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.ResultsThe Tortula chloroplast genome is ~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 ~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.ConclusionsAlthough 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.

VEGETATIVE GROWTH OF ACETABULARIA ACETABULUM (CHLOROPHYTA): STRUCTURAL EVIDENCE FOR JUVENILE AND ADULT PHASES IN DEVELOPMENT1

We characterized vegetative development in two inbred cell lines of Acetabularia acetabulum (L.) Silva. Cell growth occurred at the apex and by elongation of older interwhorls throughout vegetative development. Although cell length and hairs per whorl increased regularly during development, interwhorl length, hair persistence on the stalk, and complexity of each whorl (degree of branching of whorl hairs) showed sharp discontinuities during development in both cell lines. The first (earliest) discontinuity, formation of a short interwhorl, was the sixth interwhorl made in all cells. Even though cell line Aa1055 was twice the height ofAa4010 when mature, cells in both lines were 0.8–1.0 cm tall after formation of the short interwhorl. The second discontinuity, increases in hair persistence on the stalk and complexity of each whorl of hairs, began shortly before cap initiation. We propose the following nomenclature: 1) that slower growth before formation of the short interwhorl be called “juvenile”; 2) that more rapid growth after formation of the short interwhorl be called “adult”; and 3) that adult growth be separated into “early” and “late” phases by the discontinuities in whorl hair characteristics. The proposed developmental phases (juvenile, early adult, and late adult) are temporally sequential and spatially stacked.

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

BackgroundRecent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome.ResultsAnalysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella.ConclusionsWhen placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.

The Complete Plastid Genome Sequence of Angiopteris evecta (G. Forst.) Hoffm. (Marattiaceae)

ABSTRACT We have sequenced the complete plastid genome of the fern Angiopteris evecta. This taxon belongs to a major lineage (marattioid ferns) that, in most recent phylogenetic analyses, emerges near the base of the monilophytes. We used fluorescence activated cell sorting (FACS) to isolate organelles, rolling circle amplification (RCA) to amplify the plastid genome, followed by shotgun sequencing to 8X depth coverage, and then we assembled these reads to obtain the plastid genome sequence. The circular genome map has 153,901 bp, containing inverted repeats of 21,053 bp each, a large single-copy region of 89,709 bp, and a small single-copy region of 22,086 bp. Gene order is similar to that of Psilotum. Several unique characters are observed in the Angiopteris plastid genome, such as repeat structure in a pseudogene. We make structural comparisons to Psilotum and Adiantum plastid genomes. However, the overall structural similarity to Psilotum indicates either wholesale conservation of genome organization, or (less likely) repeated convergence to a stable structure. The results are discussed in relation to a growing comparative database of genomic and morphological characters across the green plants.

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

BackgroundAcetabularia 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.ResultsClustering 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.ConclusionsOur 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.

The Importance of Emerging Model Systems in Plant Biology

Model systems in plant biology include a range of species spanning from “well-established” to “emerging” models, depending on the degree to which they have been developed. There are two phases to building a model system: initiation and maintenance. Model species are initiated usually with a novel and often classic contribution to science (that is, they have provided insight into a process that was previously poorly understood). Mendel’s insights into genetics that came from analysis of the phenotype of pea seed coats is a good example. To be sustained, model systems must be experimentally tractable in general and also have a unique area in which their contributions are outstanding. They must be tractable in enough arenas—genetics, development, culture, transformation, and so on—so roadblocks do not prevent progress. For example, Xenopus oocytes are outstanding for localization of determinants, and the dearth of genetics can be circumvented with microinjection. Model species must be recognized by the scientific community (in print by peer review, by representation in symposia, and with funding) to emerge as a new model and then to grow into wellestablished systems. Emerging Model Systems in Plant Biology, a special issue of Journal of Plant Growth Regulation, helps to inaugurate a new format for Springer-Verlag centered on bringing together reviews on a timely topic. Why are emergent model systems for plant biology a timely topic? Indeed, why should we have more than one model plant system? We will know the complete sequence of Arabidopsis thaliana soon. This system has good genetics and excellent molecular tools (cDNA and genomic libraries, bacterial artificial chromosomes, microarrays, ESTs, and so on). Prominent scientists have voiced the opinion that progress in plant biology was slowed to a snail’s pace for years by working on too many species at one time. Clearly, having focused on A. thaliana has pushed plant biology forward by leaps and bounds in a short time. In the age of modern molecular genetics when we can clone a gene from one plant and use molecular and biochemical techniques (that is, polymerase chain reaction and antibodies) to study that same gene in another species, why bother with more than one plant model system? It is not an overstatement to say that plants— from green algae to angiosperms—represent the most diverse biochemistry, architecture, life history (including alternation of generations), reproductive biology (sexual and asexual), and body plans on Earth. Flowering plants have an estimated 300,000 species compared with only 4,500 for our closest relatives, the mammals, a group of approximately the same age. No one plant, not even Arabidopsis thaliana, can encompass this enormous diversity at the whole plant, physiologic, chemical, genetic, or molecular level. It behooves us to understand this biodiversity so that we can better use it and protect it as the population and environmental impact of our own species explodes into the next century. Mankind uses plants for fuel, building materials, clothes, medicine, decoration (including beauty products and holiday talismans), recreation, food, and drink. It is hard to imagine life without plants—indeed, plants make Online publication 9 March 2001 *Corresponding author; e-mail: mandoli@u.washington.edu J Plant Growth Regul (2000) 19:249–252 DOI: 10.1007/s003440000038

A NEW, ARTIFICIAL SEA WATER THAT FACILITATES GROWTH OF LARGE NUMBERS OF CELLS OF ACETABULARIA ACETABULUM (CHLOROPHYTA) AND REDUCES THE LABOR INHERENT IN CELL CULTURE1

We designed a new, artificial seawater (Ace 25) in order to grow bulk cultures of Acetabularia acetabulum (L.) Silva with a minimum of labor. To this end, we modified the traditional recipe for cell growth (Müllers medium as modified by Schweiger et al.) We eliminated five of the inorganic chemicals and determined the optimum concentration for 16 of the remaining 18 inorganic chemicals from modified Müllers seawater. Ace 25 enables growth of A. acetabulum from the beginning of the juvenile phase through gametangial formation in 11 weeks at high cell densities without medium replenishment. This represents a 98% reduction in the seawater volume required to mature each cell, a 30–40% reduction of the duration of the life cycle, an estimated 80% reduction in labor, and a 50–95% reduction in the space required for culturing A. acetabulum as compared with traditional procedures. These improvements may facilitate studies that require large numbers of cells such as population studies, genetics, and biochemistry, contribute to understanding the nutritional requirements of marine algae, and extend the use of this cell to those who lack the space or manpower to grow the cells in the traditional manner.

Calcification and measurements of net proton and oxygen flux reveal subcellular domains in Acetabularia acetabulum

Abstract. Vegetative adults of Acetabularia acetabulum (L.) Silva were studied as a model system for subcellular patterning in plants, and a description of several phenotypic and physiological characteristics that reveal patterns of subcellular differentiation in this unicellular macroalga was undertaken. Initially, calcification patterns were studied. Under favorable conditions, the rhizoid and most of the stalk calcified. Only the apical 10–20% of the stalk and a small region adjacent to the rhizoid remained uncalcified. Calcification in algae has been reported to result from a biologically mediated local increase in alkalinity. To test this model extracellular pH and extracellular hydrogen ion gradients were examined with ion-selective, self-referencing, electrodes. In the light, A. acetabulum displayed a general pattern of extracellular alkalinity around the entire alga, although in some individuals the region near the rhizoid and the rhizoid itself displayed extracellular acidity. Acetabularia acetabulum also displayed net hydrogen ion influx at the rhizoid and the apical half of the stalk, variable flux in the lower part of the stalk, and net hydrogen ion efflux at the base of the stalk next to the rhizoid. The lack of complete correlation between external pH patterns and calcification suggests that other factors contribute to the control of calcification in this alga. To examine whether net hydrogen ion flux patterns correlated with photosynthetic or respiration patterns, oxygen flux was measured along the stalk using self-referencing O2 electrodes. Photosynthetic oxygen evolution occurred at comparable levels throughout the stalk, with less evolution in the rhizoid. Respiration mainly occurred near and in the rhizoid, with less O2 consumption occurring more apically along the stalk. Our studies of calcification patterns, net hydrogen ion flux and O2 flux revealed several overlapping patterns of subcellular differentiation in A. acetabulum.

An analysis of morphogenesis of the reproductive whorl of Acetabularia acetabulum

Abstract.Acetabularia acetabulum (Linn.) P.C. Silva, is a useful system for studying changes in shape because it is large, morphologically complex unicell. The middle, or gametophore lobe of the cap grows radially from the stalk axis as a disc and the fully grown cap can be one of several shapes: flat, concave, convex, and saddle. The shape of the cap normally changes during the first three and a half weeks of reproductive development: individual caps within a population change shape in a stereotypical progression, with the majority proceeding from concave to flat to saddle. Marking the existing surface of caps with carbon grains revealed that the majority of growth occurs near the center, not at the perimeter, of caps. The shape of the mature cap appeared to be independent of algal height, number of gametophores per cap, and final cap diameter. Removing the rhizoid, which contains the nucleus, suggested that the contribution of the nucleus may be important for changes in shape during early cap growth. Based on these data, we present a simple model of cap shape development that suggests both differential growth and biophysical factors may contribute to the final shape of caps of A. acetabulum.