Caren Chang - US grants

Existing genefinders use information about splice sites and differences
between exons and introns, but do not explicitly incorporate information
about sequences known as splicing enhancers, which promote incorporation
of exons into mRNA. This is a project to characterize exonic splicing
enhancers in Arabidopsis. Although splicing enhancers likely function in
splice site selection in many plant genes, and contribute to the
regulation of alternative splicing, plant splicing enhancers have not
yet been described in detail. Computational analysis of a database of
Arabidopsis exons and introns will be used to identify candidate
splicing enhancer sequences. The role of these sequences, and sequences
from genes that are known to be alternatively spliced, will be tested in
transgenic Arabidopsis using a splicing reporter construct. The activity
of these enhancers will be examined in transgenes that depend on exon
inclusion for expression. This will include analysis of the
tissue-specificity of splicing enhancer activity and should provide an
extensive database of information about the role of particular sequences
in promoting splicing. This project will generate:

1) 2,000 publicly available transgenic lines, available through the
ABRC, carrying splicing reporter genes with defined candidate splicing
enhancer sequences

2) A description of marker gene expression for each splicing enhancer
candidate. This will consist of a description of all expression patterns
and images of typical and selected patterns, available through the
internet (http://www.tigr.org/2010-splicing/) and linked to the seed stocks

3) Improved genefinding and gene annotation available as improvements to
the existing GlimmerM server (http://www.tigr.org/softlab/glimmerm/).

Effective use of the complete nucleotide sequence of Arabidopsis
requires improved gene annotation. Information about the messenger RNA
products of genes is incomplete. This project will provide to the
scientific community both experimental data on splicing enhancers that
will improve gene annotation, and genetic material for further study.

This award supports the acquisition of eight plant growth chambers and computerized monitoring system that will provide the foundation of a new multi-user plant growth facility in the Department of Cell Biology and Molecular Genetics at the University of Maryland. The facility will be used by investigators whose work with the model plant Arabidopsis thaliana, but address diverse problems. These include the function of vacuolar ion pumps and ethylene receptors, the control and function of genes encoding gluconases used in cell-wall formation and remodeling, and splicing of messenger RNA. Arabidopsis has been increasingly adopted as the experimental material of choice for studies of basic plant biology because of the knowledge of the complete genomic sequence and the availability of large mutant collections, extensive databases and powerful new molecular genetic tools. These chambers will increase the growth area available to these researchers by 3 to 4-fold, and will provide precise control of day-length, light and temperature, all of which can affect the outcome of experiments intended to increase knowledge of plant development, physiology and genetics.

Land plants (or embryophytes), which include flowering plants, gymnosperms, ferns, mosses, and their relatives, are vital to almost every aspect of human life. They provide the food that we eat, absorb CO2, put oxygen in the air, provide wood, fiber, and were the original source of most coal. This project will explore the fundamental, shared properties of land plants by studying the genomes of the closest living relatives of land plants. The charophyte green algae are structurally simple organisms that share relatively recent ancestry with plants, and consequently understanding these organisms can help understand the properties of the ancestors of plants. Seven charophytes will be studied by EST analysis, with a goal of obtaining roughly 10,000 individual EST sequences from each of these seven organisms. EST analysis involves sequencing randomly-selected clones from a library made of reverse-transcribed messenger RNA, and is complementary to complete-genome sequencing. In addition to EST sequencing, the project will fully sequence a substantial fraction of the clones, will annotate and analyze the sequences for their potential significance to the evolution of land plants, and will perform preliminary studies such as genome size estimation that will provide background information needed for future genomic study.

This project will greatly expand the database of gene sequences available from the closest living relatives of land plants. This will lead to an improved understanding of the fundamental properties of land plants and their relatives. In addition, it will provide comparative data that are essential for more applied studies that seek to control and manipulate plant properties such as signal transduction - which controls plant development and environmental responses - key biochemistries such as lignification and secondary-products chemistry, and crop yield and environmental tolerance. The work will be coordinate with, and will complement, ongoing efforts to understand plant and microbial genomes, and the structure of the tree of life, and will lead toward the development of novel model systems. It will provide training for at least one postdoctoral associate and several undergraduates.

[unreadable] DESCRIPTION (provided by applicant): The broad, long-term objective is to understand the mechanisms by which organisms control transition metal ions and the roles of these metals, particularly copper, in cell signaling and regulatory pathways. Copper homeostasis is of major importance to human health, yet the pathways for intracellular copper trafficking are unclear, and relatively few components in copper sensing and trafficking have been identified. This proposal focuses on RTE, a previously undescribed gene of novel sequence, which is hypothesized to encode a sensor, transporter or chaperone for copper. The RTE gene is highly conserved, and is present in a single copy in animals and in two copies in plants. The aim of this proposal is to define the role of RTE in metal homeostasis. The studies will center primarily on the RTE1 gene of the model plant Arabidopsis thaliana. Genetic analyses in Arabidopsis have linked RTE1 to two copper-binding proteins in ethylene signal transduction. One is the copper-requiring ethylene receptor ETR1, whose function is dependent on RTE1. The other is RAN1, a homolog of the human Menkes/Wilson's P-type ATPase copper transporter, which is likewise required for ethylene receptor function. Investigating the genetic and cellular basis for these connections to RTE1 in Arabidopsis will utilize powerful genetic tools for understanding the cellular roles of RTE. Parallel experiments with respect to RTE and the Menkes/Wilson's homolog CUA-1 will be performed in the animal genetic model Caenorhabditis elegans, allowing comparison and integration of results from an animal system with those in plants. Saccharomyces cerevisiae and mammalian cell culture will be employed for ethylene binding assays and copper binding assays, respectively. The proposed experiments will use a combination of molecular genetics, cell biology and biochemical methods. The elucidation of RTE function should have a direct impact on understanding the processes of metal homeostasis for the benefit of human health. [unreadable] [unreadable]

The goal of this project is to elucidate the mechanisms that control cell fate determination in the male gametophyte of the fern Marsilea vestita. Specifically, studies will focus on the spatial and temporal regulation of translation that underlies basal body formation and nuclear remodeling in the spermatids. Spermatogenesis in this fern is a process that is activated by placing dry spores into water and involves a series of nine rapid cell division cycles in precise planes within the spore wall to produce 32 spermatids and 7 sterile cells. There is no movement of cells, so size and position within the spore wall defines cell fate. Gametophyte development takes place in the absence of new transcription; instead, the gametes are formed with proteins that were already present in the spore, or translated from mRNAs that had been transcribed and stored prior to spore desiccation. Within the spermatids, differentiation involves the formation of basal bodies in the absence of preexisting centrioles in a unique particle known as a blepharoplast. The spermatids then form an elaborate signature microtubule cytoskeleton known as the multilayered structure (MLS). The later phases of spermatid maturation involve nuclear remodeling, cell reshaping, and finally ciliogenesis, to produce a multiciliated, spirally-shaped motile gamete that looks nothing like the cell that gave rise to it. None of these events occurs in the sterile cells, though all of the cells present within the spore wall arose from the same progenitor. Thus, the male gametophyte of Marsilea provides a unique opportunity to study multiple aspects of cellular differentiation in populations of synchronously developing cells, where the cells are permeable to the entry of macromolecules at the time the spores are placed into water. The specific objectives of this project are to (1) determine how the core components of the exon junction complex affect patterns of centrin translation essential for the process of de novo basal body formation; (2) characterize the RNA-binding proteins that regulate the abundance of alpha-tubulin, which, together with centrin, affects basal body production and formation of the MLS; and (3) determine how spermidine affects temporal and spatial patterns of translation, thereby influencing cell fate specification within the gametophyte.

This project will provide research training and mentoring of undergraduate and graduate students, postdoctoral associates and visiting scholars. In addition, laboratory courses have been developed that provide training in cell biology and microscopy for undergraduate and graduate students and a website focused on an "Introduction to Mitosis" has been developed and is accessible by the public at http://www.life.umd.edu/CBMG/faculty/wolniak/wolniakmitosis.html.

Arabidopsis thaliana has been the subject of genetic analysis for more than forty years.
Currently thousands of researchers worldwide work with this highly tractable reference plant, using it to study nearly all aspects of plant biology. The plant research community has responded to the challenge of exploiting the complete genome sequence of Arabidopsis with an ambitious functional genomics program, driving the discovery of Arabidopsis gene functions and, by extension, gene functions in all plants. A key component in the synergistic interactions among Arabidopsis research laboratories is the opportunity to meet and share scientific results with colleagues from around the world at the annual International Conference on Arabidopsis Research (ICAR). The 19th annual ICAR, to be held July 23-27, 2008, in Montreal, Canada, is anticipated to draw over 1000 delegates from countries around the world. The participation of graduate students, postdoctoral associates, and beginning investigators will be fostered through travel awards to attend the conference. The funding provided by NSF will be used to broaden participation of young scientists from the US scientific community and from developing countries. Conference program and abstracts will be publicly available on the conference website, which is linked to the Arabidopsis Information Resource (TAIR) website (www.arabidopsis.org).

Intellectual merit. Ethylene is a gaseous plant hormone that has profound effects on numerous aspects of plant growth and development, including adaptive responses to a wide range of biotic and abiotic stresses. The current framework of the ethylene signal transduction pathway starts with ethylene perception and leads to changes in gene expression. Much of what is known about the ethylene signaling pathway is based on genetic dissection in the reference plant Arabidopsis thaliana. While great progress has been made in identifying key players in the signaling pathway, the molecular mechanisms by which these proteins signal remain largely unknown. A limitation is that genetic screens cannot directly detect hormone-induced changes in protein level, activity, localization or function, which form the basis of signal transduction. In addition, a number of components may be recalcitrant to genetic dissection. This project has two objectives. The first is to attain new levels of understanding of ethylene signal transduction using proteomic methods to identify previously unknown ethylene signaling components and their molecular mechanisms. These methods will identify proteins that are rapidly modified in response to ethylene, as well as proteins that physically interact in the pathway. Protein modification and protein-protein interactions are essential to the mechanisms of intracellular signal transduction, but have been relatively unexplored in ethylene signaling. The second objective is to carry out analyses of mutants and genes that are currently in hand, with a particular focus on a gene called RTE1, which regulates signaling by the ETR1 ethylene receptor. RTE1 is a novel membrane protein conserved in plants, animals and some protists. The cellular role of RTE1 and how RTE1 specifically regulates ETR1 signaling will be investigated through a combination of molecular genetics, cell biology and biochemistry approaches. The extensive molecular genetic tools that exist for ethylene signaling, coupled with the availability of powerful proteomic methods, provide an exceptional opportunity to advance our knowledge of ethylene signal transduction.

Broader impacts. This project provides research training and mentoring of undergraduates, graduate students, and a postdoctoral researcher, who are typically from underrepresented groups. Laboratory members will present their research at a variety of scientific meetings, both local and international, and will participate in teaching outreach. The postdoctoral researcher will follow a career development plan supported by research training in the laboratory, student mentoring experiences and professional development seminars and workshops. Given the fundamental importance of ethylene in plant growth and development, the mechanistic insights provided by the proposed studies could have an impact on enhancing agricultural products for human nutrition and plant biomass optimization.

Collaborative grants have been awarded to the University of Maryland, the University of Iowa and St. Bonaventure University to develop a methodology that exploits the wealth of annotation knowledge, notably Gene Ontology (GO) and Plant Ontology (PO) annotations of Arabidopsis genes. Motivated by the availability of rich and as yet insufficiently tapped collections of gene annotations, the project aims to facilitate the discovery of hidden knowledge that could be the basis of further scientific research. The methodology will extract patterns of interest from annotation graphs (pattern discovery). Literature-based methods will extract sentences that validate the biological meaning underlying these patterns (pattern validation). To demonstrate the methodology, the PattArAn tool (Patterns in Arabidopsis Annotations) will be customized for Arabidopsis. PattArAn will provide the user with a graphical presentation of patterns of Arabidopsis genes and associated GO and PO CV terms. Graph data mining techniques and efficient algorithmic solutions to identify dense subgraphs (DSG) and to perform graph summarization (GS) will be developed. Algorithms to mine the literature for relevant sentences for an extracted pattern (referred to as the imprint) will be developed. PattArAn will enable iterative exploration and will incorporate allied steps such as consulting gene function prediction. The project will involve collaboration with biologists for building and refining annotation graphs, and validating patterns to ensure relevance to their research.

The project makes broad contributions to the Arabidopsis thaliana community. PattArAn may assist Arabidopsis curators to manage GO-PO annotations and complement existing tools such as Textpresso and AraNet. It can also be used to bootstrap an annotation database for other plant species given that their genome sequence information is available. The project offers significant research and educational experiences for graduate students (University of Maryland and Iowa) and undergraduate students (St. Bonaventure University). Team members will continue to mentor women and students from under-represented communities, participate in outreach activities, lead a Journal Club, etc. The outcomes from this research project will be disseminated via biology and bioinformatics venues. More information may be obtained at the project website: https://wiki.umiacs.umd.edu/clip/pattaran/.

Intellectual Merit: Cellular calcium mediates diverse processes in both animal and plant cells, including gene expression, cell proliferation, cell division, metabolism, and cell movement. Stomata are microscopic pores each formed by a pair of guard cells that shrink and swell to regulate respiration. Calcium channels in the plasma membrane of these specialized cells play a crucial role in allowing an increase of cytosolic Ca2+, which causes these pores to close thus limiting water loss by the plant. Patterns of cytosolic Ca2+ dynamics encode information that impacts stomatal behavior and controls the specificity and efficiency of gene expression; however, the molecular identity of these Ca2+ channels in plants remains elusive. In addition, molecular mechanisms by which these Ca2+ patterns regulate cellular responses, including gene expression, have yet to be identified. Using Arabidopsis guard cells as a model system, the aim of this project is to investigate how a cellular Ca2+ signal is transduced by molecular components at the single-cell level. In this project, a multidisciplinary approach that includes computational modeling and molecular genetic analysis, will be used to characterize a family of putative Ca2+-permeable channel proteins in the plasma membrane and to analyze how cytosolic Ca2+ dynamics modulates gene expression in guard cells and contributes to the regulation of stomatal behavior. The results of this research will reveal novel molecular components of cellular Ca2+ signaling and provide new insights into Ca2+ dynamics-controlled cellular responses in plants.

Broader Impacts: Fresh water scarcity is one of the major global problems this century, and 65% of global fresh water is used for agriculture. Plants lose over 95% of their water via transpiration through stomatal pores in the leaf surface. Since cellular calcium plays a key role in the regulation of stomatal behavior, results and knowledge arising from the proposed research will contribute to protecting the environment and to improving agricultural productivity. Two postdoctoral fellows, two graduate students and one or more undergraduate students will participate in this project. The PI and co-PI will provide extensive mentoring to prepare postdoctoral associates for their future careers. The PI and co-PI will continue making a commitment to broadening the participation of students from underrepresented groups. The PI and co-PI will give presentations and lectures to the public to stimulate general interest in science and science education. In collaboration with colleagues, the PI plans to offer an open house to local and state governmental officials to provide a non-technical explanation of our research and give these policymakers an opportunity to see first hand tax dollars at work.

Plants produce hormones such as ethylene that collectively control essentially all aspects of plant biology. This project advances a new hypothesis that a small molecule, which plants use to make ethylene, functions itself as a novel plant hormone that impacts plant growth and development, and further investigates how the hormone functions at the molecular level throughout the evolutionary breadth of plants. The project has the potential to transform the plant hormone field by altering existing dogma, by requiring the reevaluation of several decades of ethylene literature and by impacting how future ethylene experiments are carried out. The knowledge gained from this project could contribute to future strategies for modifying crop plants for the benefit of society. The project broadens participation of underrepresented students by providing two to three student internships per year in the lab of the principal investigator. The internships involve partnerships with Howard University (an Historically Black College or University) and Eleanor Roosevelt High School (a local public school in which over 70% of the students are underrepresented minorities). The interns carry out aspects of the project while receiving valuable training in scientific methods, data analysis and communication. The project also provides training and career preparation for two postdoctoral scientists, two graduate students and two University of Maryland undergraduates. To further broaden participation, the principal investigator co-teaches summer research workshops for Howard University undergraduates.

The well-known ethylene precursor in the ethylene biosynthesis pathway is 1-aminocyclopropane-1-carboxylic acid (ACC), a non-proteinogenic amino acid. Compelling new data suggest that ACC itself may be an important signaling molecule that evolutionarily predated the ability of higher land plants to efficiently convert ACC to ethylene. In particular, ACC inhibits cellular differentiation and growth in the liverwort Marchantia polymorpha. This project tests the hypothesis that ACC is a novel plant hormone through analyses of ACC function, including an investigation of ACC signaling mechanisms. Insight into ACC function is provided by studies of ACC synthesis mutants and ACC responses in Marchantia, and in evolutionarily relevant species in the plant lineage (Chlamydomonas reinhardtii, Spirogyra pratensis, Physcomitrella patens and Arabidopsis thaliana), thus addressing the conservation of ACC function, while taking advantage of lower gene copy number in basal plants. In Arabidopsis, preliminary findings indicate that ACC can induce pollen tube growth concomitant with Ca2+ influx and can stimulate primary root growth. ACC activates Ca2+ currents in Arabidopsis root protoplasts, and notably, this activation is dependent on glutamate receptor-like (GLR) ionotropic channels. The project tests the hypothesis that ACC is a GLR ligand using a combination of molecular genetics, patch-clamping, ion-specific vibrating probes and live imaging of Ca2+. Genetic evidence is sought based on phenotypic comparisons of glr mutants and ACC synthesis mutants in the range of plant species. The activation of GLRs by ACC, leading to the regulation of Ca2+ signaling in pollen tube growth and/or root growth, would be a breakthrough in understanding mechanisms of ACC signaling, as well as GLR signaling, and would represent a ligand-operated system of Ca2+ regulation for which no consensual system currently exists in plants. The striking ACC response uncovered in Marchantia provides for a genetic screen for ACC signaling mutants, followed by gene cloning based on T-DNA tagging or whole genome sequencing.