Eric D. Brenner

Cycads: evolutionary innovations and the role of plant-derived neurotoxins

Cycads are an important relic from the past and represent the oldest living seed plants. Cycads have been instrumental in our understanding the evolution of angiosperms and gymnosperms because they have recognizable morphological characteristics intermediate between less-recently evolved plants such as ferns and more-derived (advanced) plants including the angiosperms. Cycads also produce several compounds that are carcinogenic and neurotoxic. Because of their unique placement in terrestrial plant evolution, molecular studies should help to define the origins of structures that led to the rise of seed plants and the role of neurotoxic compounds that are found in cycads.

The Impact of Outgroup Choice and Missing Data on Major Seed Plant Phylogenetics Using Genome-Wide EST Data

Background Genome level analyses have enhanced our view of phylogenetics in many areas of the tree of life. With the production of whole genome DNA sequences of hundreds of organisms and large-scale EST databases a large number of candidate genes for inclusion into phylogenetic analysis have become available. In this work, we exploit the burgeoning genomic data being generated for plant genomes to address one of the more important plant phylogenetic questions concerning the hierarchical relationships of the several major seed plant lineages (angiosperms, Cycadales, Gingkoales, Gnetales, and Coniferales), which continues to be a work in progress, despite numerous studies using single, few or several genes and morphology datasets. Although most recent studies support the notion that gymnosperms and angiosperms are monophyletic and sister groups, they differ on the topological arrangements within each major group. Methodology We exploited the EST database to construct a supermatrix of DNA sequences (over 1,200 concatenated orthologous gene partitions for 17 taxa) to examine non-flowering seed plant relationships. This analysis employed programs that offer rapid and robust orthology determination of novel, short sequences from plant ESTs based on reference seed plant genomes. Our phylogenetic analysis retrieved an unbiased (with respect to gene choice), well-resolved and highly supported phylogenetic hypothesis that was robust to various outgroup combinations. Conclusions We evaluated character support and the relative contribution of numerous variables (e.g. gene number, missing data, partitioning schemes, taxon sampling and outgroup choice) on tree topology, stability and support metrics. Our results indicate that while missing characters and order of addition of genes to an analysis do not influence branch support, inadequate taxon sampling and limited choice of outgroup(s) can lead to spurious inference of phylogeny when dealing with phylogenomic scale data sets. As expected, support and resolution increases significantly as more informative characters are added, until reaching a threshold, beyond which support metrics stabilize, and the effect of adding conflicting characters is minimized.

EST analysis in Ginkgo biloba: An assessment of conserved developmental regulators and gymnosperm specific genes

BackgroundGinkgo biloba L. is the only surviving member of one of the oldest living seed plant groups with medicinal, spiritual and horticultural importance worldwide. As an evolutionary relic, it displays many characters found in the early, extinct seed plants and extant cycads. To establish a molecular base to understand the evolution of seeds and pollen, we created a cDNA library and EST dataset from the reproductive structures of male (microsporangiate), female (megasporangiate), and vegetative organs (leaves) of Ginkgo biloba.ResultsRNA from newly emerged male and female reproductive organs and immature leaves was used to create three distinct cDNA libraries from which 6,434 ESTs were generated. These 6,434 ESTs from Ginkgo biloba were clustered into 3,830 unigenes. A comparison of our Ginkgo unigene set against the fully annotated genomes of rice and Arabidopsis, and all available ESTs in Genbank revealed that 256 Ginkgo unigenes match only genes among the gymnosperms and non-seed plants – many with multiple matches to genes in non-angiosperm plants. Conversely, another group of unigenes in Gingko had highly significant homology to transcription factors in angiosperms involved in development, including MADS box genes as well as post-transcriptional regulators. Several of the conserved developmental genes found in Ginkgo had top BLAST homology to cycad genes. We also note here the presence of ESTs in G. biloba similar to genes that to date have only been found in gymnosperms and an additional 22 Ginkgo genes common only to genes from cycads.ConclusionOur analysis of an EST dataset from G. biloba revealed genes potentially unique to gymnosperms. Many of these genes showed homology to fully sequenced clones from our cycad EST dataset found in common only with gymnosperms. Other Ginkgo ESTs are similar to developmental regulators in higher plants. This work sets the stage for future studies on Ginkgo to better understand seed and pollen evolution, and to resolve the ambiguous phylogenetic relationship of G. biloba among the gymnosperms.

ESTimating plant phylogeny: lessons from partitioning

BackgroundWhile Expressed Sequence Tags (ESTs) have proven a viable and efficient way to sample genomes, particularly those for which whole-genome sequencing is impractical, phylogenetic analysis using ESTs remains difficult. Sequencing errors and orthology determination are the major problems when using ESTs as a source of characters for systematics. Here we develop methods to incorporate EST sequence information in a simultaneous analysis framework to address controversial phylogenetic questions regarding the relationships among the major groups of seed plants. We use an automated, phylogenetically derived approach to orthology determination called OrthologID generate a phylogeny based on 43 process partitions, many of which are derived from ESTs, and examine several measures of support to assess the utility of EST data for phylogenies.ResultsA maximum parsimony (MP) analysis resulted in a single tree with relatively high support at all nodes in the tree despite rampant conflict among trees generated from the separate analysis of individual partitions. In a comparison of broader-scale groupings based on cellular compartment (ie: chloroplast, mitochondrial or nuclear) or function, only the nuclear partition tree (based largely on EST data) was found to be topologically identical to the tree based on the simultaneous analysis of all data. Despite topological conflict among the broader-scale groupings examined, only the tree based on morphological data showed statistically significant differences.ConclusionBased on the amount of character support contributed by EST data which make up a majority of the nuclear data set, and the lack of conflict of the nuclear data set with the simultaneous analysis tree, we conclude that the inclusion of EST data does provide a viable and efficient approach to address phylogenetic questions within a parsimony framework on a genomic scale, if problems of orthology determination and potential sequencing errors can be overcome. In addition, approaches that examine conflict and support in a simultaneous analysis framework allow for a more precise understanding of the evolutionary history of individual process partitions and may be a novel way to understand functional aspects of different kinds of cellular classes of gene products.

Nuclear genome size in Selaginella

Estimates of nuclear genome size for 9 Selaginella species were obtained using flow cytometry, and measurements for 7 of these species are reported for the first time. Estimates range from 0.086 to 0.112 pg per holoploid genome (84-110 Mb). The data presented here agree with the previously published flow cytometric results for S. moellendorffii. Within the 9 species sampled here, chromosome number varies from 2n = 16 to 2n = 27. Nuclear genome size appears to be strongly correlated with chromosome number (Spearmans rank correlation; p = 0.00003725). Cultivated S. moellendorffii lacks sexual reproduction--manifest by the production of abortive megasporangia. Flow cytometric data generated from a herbarium specimen of a fertile wild-collected S. moellendorffii are virtually indistinguishable from the data generated from fresh material (0.088 vs. 0.089 pg/1C). Therefore, the limited fertility observed in cultivated plants is probably not the result of abnormal chromosome number (e.g., induced by interspecific hybridization).

When North and South don’t mix: genetic connectivity of a recently endangered oceanic cycad, Cycas micronesica, in Guam using EST‐microsatellites

Subject to environmental changes and recurrent isolation in the last ca. 250 Ma, cycads are often described as relicts of a previously common lineage, with populations characterized by low genetic variation and restricted gene flow. We found that on the island of Guam, the endemic Cycas micronesica has most of the genetic variation of 14 EST‐microsatellites distributed within each of 18 genetic populations, from 24 original sampling sites. There were high levels of genetic variation in terms of total number of alleles and private alleles, and moderate levels of inbreeding. Restricted but ongoing gene flow among populations within Guam reveals a genetic mosaic, probably more typical of cycads than previously assumed. Contiguous cycad populations in the north of Guam had higher self‐recruitment rates compared to fragmented populations in the south, with no substantial connection between them except for one population. Guam’s genetic mosaic may be explained by the influence of forest continuity, seed size, edaphic differences, and human transport of cycads. Also important are the extent of synchrony among flushes of reproductive female seed‐bearing sporophylls and restricted pollen movement by an obligate mutualist and generalist insects. An NADH EST‐locus under positive selection may reflect pressure from edaphic differences across Guam. This and three other loci are ideal candidates for ecological genomic studies. Given this species’ vulnerability due to the recent introduction of the cycad aulacaspis scale, we also identify priority populations for ex situ conservation, and provide a genetic baseline for understanding the effects of invasive species on cycads in the Western Pacific, and islands in general.

A mutation in the Proteosomal Regulatory Particle AAA - ATPase-3 in Arabidopsis impairs the light-specific hypocotyl elongation response elicited by a glutamate receptor agonist, BMAA

BMAA is a cycad-derived glutamate receptor agonist that causes a two- to three-fold increase in hypocotyl elongation on Arabidopsis seedlings grown in the light. To probe the role of plant glutamate receptors and their downstream mediators, we utilized a previously described genetic screen to identify a novel, BMAA insensitive morphology (bim) mutant, bim409. The normal BMAA-induced hypocotyl elongation response observed on wild-type seedlings grown in the light is impaired in the bim409 mutant. This BMAA-induced phenotype is light-specific, as the bim409 mutant exhibits normal hypocotyl elongation in etiolated (dark grown) plants (+ or − BMAA). The mutation in bim409 was identified to be in a gene encoding the ProteosomalRegulatory Particle AAA-ATPase-3 (RPT3). Possible roles of the proteosome in Glu-mediated signaling in plants is discussed.

Origin of a novel regulatory module by duplication and degeneration of an ancient plant transcription factor.

It is commonly believed that gene duplications provide the raw material for morphological evolution. Both the number of genes and size of gene families have increased during the diversification of land plants. Several small proteins that regulate transcription factors have recently been identified in plants, including the LITTLE ZIPPER (ZPR) proteins. ZPRs are post-translational negative regulators, via heterodimerization, of class III Homeodomain Leucine Zipper (C3HDZ) proteins that play a key role in directing plant form and growth. We show that ZPR genes originated as a duplication of a C3HDZ transcription factor paralog in the common ancestor of euphyllophytes (ferns and seed plants). The ZPRs evolved by degenerative mutations resulting in loss all of the C3HDZ functional domains, except the leucine zipper that modulates dimerization. ZPRs represent a novel regulatory module of the C3HDZ network unique to the euphyllophyte lineage, and their origin correlates to a period of rapid morphological changes and increased complexity in land plants. The origin of the ZPRs illustrates the significance of gene duplications in creating developmental complexity during land plant evolution that likely led to morphological evolution.

An undergraduate study of two transcription factors that promote lateral root formation

We present a lab that enables students to test the role of genes involved in the regulation of lateral roots growth in the model plant Arabidopsis thaliana. Here, students design an experiment that follows the effects of the hormone auxin on the stimulation of genes involved in the formation of lateral root initials. These genes, known as lateral organ boundary domain containing protein (LBD) genes, are upregulated in the presence of auxin as part of a multistep molecular and biochemically controlled pathway. Depending on which LBD gene is tested, and the stage of root development, expression patterns are localized in a discrete and punctate fashion at the site of lateral root initials (LBD33), or reveal a broader localization pattern (LBD16). Students view expression using the reporter gene GUS (beta‐glucuronidase). Before GUS staining, students view root growth in a “pseudo‐aseptic” agar‐based environment that allows complete visualization of whole root development to determine the proper stage to test molecular expression.

Using Genomics to Study Evolutionary Origins of See ds

Today’s gymnosperms are relicts from groups of plants whose history extends to nearly 300 million years ago when seeds first evolved. Consequently gymnosperms have been studied in relation to the fossil record to uncover important clues to understand the appearance and evolution of seeds and pollen. Extant gymnosperms are restricted to four surviving orders: the Gnetales, Coniferales, Ginkgoales and Cycadales, providing a marvelous biological source of data which has only recently been examined at the molecular level. Efforts to understand the molecular evolution of the gymnosperms has examined the expression of gymnosperms genes with similarity to genes in angiosperms involved in the development of leaves and reproductive structures via a degenerate based primer approach. Lately, genomic approaches have been engaged to understand what genes are involved in the evolution and development of gymnosperm structures. Below is a description of genomic and molecular studies currently to understand evolution in the gymnosperms. 1. THE RISE OF THE SEED PLANTS The emergence of the seed plants is among one of the most significant developments to occur in the evolution of terrestrial plants (FOSTER and GIFFORD 1974)). Through dramatic changes in reproductive strategies, the seed plants (spermatophytes), which include the angiosperms and gymnosperms, were able to overcome one of the key barriers to reproductive efficiency. In non-seed plants, such as ferns, the gametophyte is photosynthetic, free living (exosporic) and not vascularized. Consequently fertilization can only occur in wet environments where flagellated gametes can swim from their source, the antheridia, to the archegonia to fertilize the egg. Because of their dependence on water mediated fertilization, hybridization in non-seed plants can occur only between close neighboring individuals as swimming sperm can only travel short distances. In contrast to non-seed plants, the seed plant megametophyte is retained within the spore wall (endosporic) and also within the maternal tissue. That is the megagametophyte is contained within the diploid integuments to form the ovule (Figure 1). Gamete transfer does not require water as a medium but rather it is the male gametophyte that is transferred. The male microgametophyte in seed plants is transported as a desiccation-resistant pollen grain directly to the ovule (or ovule enclosing carpel), often many miles away. Upon reaching the female reproductive structure, a tube grows from the pollen, subsequently bringing the male gamete to the female gametophyte and ultimately to the egg. Similar to the pollen grain, the female gametophyte is also dessication C. G. Williams (ed.), Landscapes, Genomics and Transgenic Conifers, 85-106. resistant via protection by diploid tissue, the nucellus and integuments. The megagametophyte is no longer free-living, or photosynthetic and must receive nutrients from the vascularized, sporophytic parent during its development. The megagametophyte remains dependent on the maternal parent during pollination and later while the pollen tube is growing prior to fertilization. In most cases fertilization occurs while the megagametophyte is attached to the sporophyte, while in the oldest surviving seed plants fertilization occurs after the ovule has been shed, such as in cycads and gingko. Upon fertilization the ovule becomes a seed. Within the seed, the megagametophyte function has been modified to serve primarily as nutrient storage tissue to sustain the developing embryo (In the angiosperms nutritive tissue typically comes from other tissue types including endosperm and cotyledons, while in gymnosperms nutrition primarily comes from the gametophyte). Upon maturity the seed becomes dehiscent and subsequently transported (by biotic or non-biotic sources) to a new locale with the possibility of finding favorable growing conditions. Consequently the mature seed consists of three generations: the maternal sporophyte, the gametophyte and the new embryonic sporophyte. Thus the ovule/seed is a major developmental innovation that enables long distance fertilization as well as dispersal of a diaspore containing both the embryo and storages tissue for its establishment in a process unique in land plants. In addition to their important role enabling long distance fertilization, seeds are fundamentally important for humans. Seeds provide food, fiber and industrial goods. Considering their importance to society, a better understanding of the origins of seeds requires innovative studies to explain seed evolution. The oldest surviving seed plants are the gymnosperms, which exhibit a number of plesiomorphic (ancient) conditions reminiscent of the early fossil seed plants. Gymnosperms are hypothesized to have descended from the progymnosperms (ROTHWELL 1988), which date back in the fossil record to approximately 285-350 MYA (ROTHWELL and SCHECKLER 1988) to 359-385 MYA (TAYLOR and TAYLOR 1993). One theory contends that the original gymnosperms were the seed ferns (FOSTER and GIFFORD 1974). The last common ancestor of extant seed plants and extant ferns is estimated by molecular clocks to have existed approximately 275-290 MYA (SAVARD et al., 1994). When molecular clock estimates include fossil calibration points, gymnosperms age ranges from 345 to 360 MYA depending on whether lycophyte or gymnosperm fossils, respectively, are used as calibration points estimations exists spanning nearly 100 million years. The discovery of new fossils from extinct gymnosperms and expanded molecular clock studies with deeper sequencing initiatives should help narrow this estimation gap. 1.1. Today’s gymnosperms: remnants of early seed plants Conflicting results between morphological and molecular data sets have made it difficult to define the precise phylogenetic hierarchy of the four extant gymnosperm clades: the Gnetales, Coniferales, Ginkgoales and Cycadales (DOYLE 1998; MAGOLLON and SANDERSON 2002; RYDIN et al., 2002). Among the gymnosperms, (SOLTIS et al., 2002b). Thus, considerable variation in gymnosperm age 86 E.D. BRENNER AND D. STEVENSON