Nina Fedoroff - US grants

The overall objective of this project is to identify genes that work together when plants respond to and develop tolerance for environmental stress. Although gene networks have been treated theoretically, biologists have never before been in a position to define the components of a network. Microarray technology, together with the increasing availability of cDNA and genome sequences, now makes it possible to identify groups of genes whose expression patterns change coordinately. However this technology is still in need of development to make it a more sensitive detection system that is also economically accessible to individual academic investigators. This team of investigators plans to develop new types of highly sensitive nucleic acid microarray detection systems, using colloidal gold particles to amplify detection of nucleic acids on a gold surface. While the overall concept represents a new approach with potential for both high sensitivity and low-cost detection, parts of each component have been demonstrated. This project will combine these elements and test two different detection schemes; one based on gold colloid-enhanced microwave mixing and the other on gold colloid-enhanced surface plasmon resonance.

The initial biological focus will be on genes whose expression levels are altered by biotic and abiotic stress. The specific stressors to be used in creating a model system for evaluating the new microarray detection schemes are ozone and pathogen attack. These stressors are the focus because of the parallels between some aspects of plant responses to ozone and to pathogens. Ozone can evoke formation of rapid necrotic lesions that mimic the pathogen-induced hypersensitive response. However, the response to both ozone and pathogens depends on the mode and duration of exposure, as well as the genetic constitution of the plant. Microarray technology will make it possible to analyze the differences and similarities between these physiological stress responses at the molecular level. This will permit the identification of gene sets whose activation is common to the stressors, as well as gene sets whose activation is unique to each. The results will facilitate studies on developmental, tissue-specific, and temporal differences in molecular stress response patterns and the combinatorial effects of multiple simultaneous stresses. It is well known that there are genetic differences among plants in the capacity to develop tolerance to stress and resistance to pathogen attack, that tolerance can develop in response to stress and that the ability to develop tolerance varies with age. The proposed experiments will pave the way for identifying and analyzing gene sets required for the development of stress tolerance and pathogen resistance.

A Microarray Facility is being developed at the Pennsylvania State University. This will initially make use of existing technology for DNA chip construction and two-color fluorescent probe hybridization. The microchip arrayer will then be used to create microarrays of PCR-amplifled cDNAs on a gold surface. Current fluorescence detection techniques will be used to develop a baseline for evaluating the new detection techniques. The broadest possible range of potential members of stress-response gene networks will be used for the construction of microarrays, beginning with candidate genes selected from results of published studies, as well as database searches for homologs. Existing Arabidopsis ESTs will be screened for additional candidate differentially expressed genes through the Monsanto Arabidopsis microarray program.

Although Arabidopsis will be used in these studies, the proposed research will be directly relevant to agronomically important plants because of the high degree of protein sequence conservation among different species. Moreover, there is a high probability that gene networks identified in Arabidopsis will have counterparts in other plants, facilitating their identification and, eventually, their manipulation to improve agronomic traits. The microarray research will benefit plant researchers by reducing cost and increasing the availability of the technology.

Dissection of biochemical functions and developmental pathways by mutating genes is central to functional genomics. Genes can be mutated by nucleotide sequence changes, insertions or deletions. Insertional mutagenesis with T-DNA and transposons has become the most widely used method for mutating plant genes. By contrast, homology-dependent gene targeting, a powerful technique that permits precise changes to be made in either coding or regulatory sequences, has not yet become a useful technique in plants. A number of factors have been identified that may contribute to the lack of gene targeting success or which can be optimized to enhance the frequency and increase the ease of detecting homology-based recombinational events in plants. This information has been used to design a new gene targeting strategy for plants. This project utilizes a high-risk strategy, however if successful, it will lead to the rapid development of a practicable system for homologous gene targeting in plants, which will have a very high pay-off in plant research. The strategy for plant gene targeting seeks to 1) enhance the probability of identifying homology-based interactions by detecting both gene conversion and recombination, 2) minimize illegitimate recombination associated with both naked DNA and T-DNA transformation, 3) make use of the observation that double-stranded DNA breaks stimulate recombination in plants and 4) enrich for plants in which the recombination donor molecule has been released from the chromosome. Because evidence has already been obtained indicating that all of the sub-components of the present strategy work in plants, putting the whole system together and testing it is the next step. The results of these experiments will provide the preliminary data for subsequent research to study molecular mechanisms in plant recombination and to identify favorable genetic backgrounds for gene targeting.

Plant growth and development, as well as plant responses to stress, are orchestrated by several hormones. The action of the hormones within the plant and within individual cells depends on proteins that transmit the hormone signals to evoke both biochemical and transcriptional changes. This project has the objective of analyzing an Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones simultaneously. Preliminary evidence suggests that the mutant gene encodes a nuclear protein that binds to double-stranded RNA. Mutant plants are short, slow to flower, have curled leaves, produce few seeds, and have slow-growing roots that do not respond to gravity normally. Mutant plants show a reduced sensitivity to the auxin and cytokinin hormones and are hypersensitive to the hormone abscisic acid (ABA). Genetic and molecular experiments in the project are designed to understand how the protein encoded by the HYL1 gene participates in the plant's perception and response to different hormones. The importance of these experiments is that they will contribute to our understanding of how plants coordinate their responses to the multiple hormone signals that impinge on their cells. Although the experiments are being performed in the model plant Arabidopsis, knowledge gained in such a system can be transferred readily to crop plants because higher plants use the same or similar systems to regulate growth and stress responses. Such knowledge will be very important in future efforts to improve the ability of agricultural plants to maintain productivity under adverse conditions.

Abstract: Mark Guiltinan (#0215923)

A grant has been awarded to Pennsylvania State University under the direction of Dr. Mark Guiltinan to develop a Plant Growth Facility consisting of six growth chambers and a small greenhouse. The facility center will provide a physical home for the graduate programs, and an organizing role for the enhancement of Plant Sciences research, teaching and outreach programs.
The objectives of the center will be to: provide state-of-the-art research facilities for a core group of resident plant scientists, including current faculty and new hires; provide collaborative space in the new building to members of the Plant Science Center; develop and enhance collaborative interactions among plant scientists and with researchers
in other fields, at Penn State and elsewhere; improve the Plant Physiology and Ecological and Molecular Plant Physiology graduate education programs, and; develop outreach programs for the general public and for pre-college students.

This center will support research in plant sciences for diverse studies, but will be particularly important to the growing number of plant scientists using the model plant species, Arabidopsis thaliana. The completion of the Arabidopsis genome sequence, along with major new funding programs in plant genomics at the NSF and USDA,
have brought us to a new era in plant research, requiring high throughput facilities for functional analysis of genes.

Penn State has made a priority effort to enhance the life sciences; enhancement of the Plant Sciences Center will help us move forward on this path, by providing necessary facilities and maximizing laboratory space in the main building. This will in turn help to attract top faculty candidates who will be concerned about availability of such facilities. The facility will provide for the first time, an interdisciplinary, inter-college facility for high quality environmental growth of plants to support the many plant scientists at Penn State. It will also serve our teaching and outreach programs, providing a facility available to all for the growth of plants.

Deletional mutagenesis has not been used as extensively as insertional mutagenesis in Arabidopsis because large deletions are often not transmitted through gametes or are lethal early in development. The investigator will carry out a systematic study of small deletions by controlling their chromosomal location, developmental timing, and parent of origin. She will make deletions of known sizes using the site-specific bacteriophage P1-encoded Cre recombinase to delete chromosomal segments between a loxP-containing T-DNA donor site and a nearby loxP-containing transposon. Using an already tested transposon "launching pad" containing loxP recognition sites for the Cre recombinase, she will identify 3-4 transposons reinserted at different distances ranging from a few kilobases to a few megabases from each of several transposon donor sites to carry out these studies. She will use Cre recombinase genes expressed from a constitutive plant promoter, as well as from a chemically inducible promoter. She will detect deletions (and inversions) by both PCR-based and genetic methods. She will determine the genetic transmissibility of deletions and analyze the development of plants in which deletions are induced at different times after germination. This work will both increase the understanding of the detrimental effects of deletions and deletional heterozygosity and provide methods to control the size, chromosomal location, and timing of deletions.
The second objective is to lay the groundwork for development of a transposon-based method to target genes to their original chromosomal locations. This technique, which the investigator calls gene "homing" to distinguish it from homology-based gene targeting, will use site-specific recombination to target a promoter-reporter cassette to a transposon-disrupted gene. If successful, this work will make it possible for the first time to study precise alterations in a plant gene's regulatory sequences in the gene's original chromatin context. The gene homing method will use transposons to target genes to their original chromatin environment by replacing a loxP-bracketed marker gene on the transposon with a promoter-reporter gene cassette. She will use existing plants with loxP transposons and transposon launching pads to determine whether an efficient "cassette replacement" technique developed in animal cells can also be used to integrate DNA segments efficiently between pairs of loxP sites in Arabidopsis. This work will establish the feasibility of replacing a transposon-borne marker gene cassette with a cassette carrying a reporter gene driven by the promoter of the disrupted gene.
This work will provide basic information and techniques that will be widely applicable in contemporary plant genomic research. Both the constructs that will be made and the techniques that will be developed will be useful in other plants.

It has recently been discovered that tiny RNA molecules, called microRNA (miRNA), are important in the development of both plants and animals, but little is yet known about how this newly-discovered regulatory mechanism operates. The principal investigator's laboratory has identified a gene in the well-characterized experimental plant Arabidopsis thaliana called Hyponastic leaves 1 (HYL1) that encodes a double-strand RNA-binding protein. Preliminary experiments suggest that the HYL1 protein participates in miRNA regulation. Levels of several miRNAs are reduced in a hyl1 mutant and elevated in a plant that overexpresses HYL1. Conversely, levels of several mRNAs predicted to be regulatory targets of the miRNAs examined are elevated in the mutant and reduced in HYL1 over-expressing plants, suggesting that the HYL1 protein is involved in either production of the miRNAs or in miRNA-mediated regulation of mRNA levels. The hyl1 mutant has a pleiotropic hormonal phenotype, exhibiting reduced sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid. The objective of this project is to determine whether and how the HYL1 protein functions in miRNA regulation of gene expression. Proteins that interact with HYL1 will be identified, located in cells, and their effect on the production of miRNAs and mRNA stability determined. How this regulatory mechanism functions in the hormonal regulation of gene expression and what kinds of signaling mechanisms activate it will also be determined. The principal investigator has been a leader in providing training opportunities for women and underrepresented minorities, both in her lab and more widely at her institution. She is also very active in communicating science issues to the general public. The scientific results may have practical applications in biotechnology.

Contemporary experimental biologists have access to a large body of disparate kinds of information about many different organisms and biological systems. Although much of the information is still in papers published in journals, primary data increasingly reside in electronically accessible databases. The volume and complexity of the available data exceed the synthetic and reasoning capacities of any individual researcher, stimulating the development of knowledge bases, which represent information about biological systems at a higher level of abstraction than do databases. A serious limitation on the usefulness of existing knowledge bases is that knowledge about the system is effectively "frozen" as it is archived. Observations that contradict the bulk of available evidence are generally omitted at the time of annotation. However, a deeper understanding of how biological systems operate often begins with an observation that contradicts existing knowledge.

The overall objective of this project is to create computational tools that allow the experimental biologist to explore accumulated information about biological systems without precluding access to contradictory information. The approach differs markedly from those currently used to construct knowledge bases, such as the pathway databases Reactome (www.reactome.org) and BioCyc (www.biocyc.org), which store currently accepted models based on expert input and literature information and they must be revised and rewritten as the knowledge grows. The approach being taken here is to instead store the ingredients for model building at the evidence level and enable biologists to continuously and actively participate in model- building through the familiar device of formulating and testing hypotheses. The hypotheses themselves are used to query the stored data by breaking each hypothesis down into its constituent relationships and extracting all of the explicit and implicit assertions that must hold in order for the hypothesis to be valid. A set of evaluation rules is applied to test these assertions for agreement with different types of data and present the user with links to information and data that support the hypothesis, as well as links to those that contradict it. Thus, static conclusions are not archived, but instead the data required are stored to elucidate relationships that exist in the system. Although the experimenter's ideas about relationships are tested against what is known, conclusions are not imposed. Rather, both supporting information and contradictions are reported and the task of evaluating the weight and significance of each left to the experimenter.

This work has several broader impacts. The importance of this approach is that it is a "thinking" tool for the experimental biologist, as opposed to a knowledge base, which is essentially an electronic textbook. Few computer assisted thinking tools are available at present and the success of this project could revolutionize how experimental biologists work. The tools will be integrated into the operation of The Arabidopsis Information Resource (TAIR: www.arabidopsis.org), a community database with 14,000 regular users world-wide.

Plants react to chemical and biological challenges from the environment with rapid changes in biochemical processes and gene expression. These responses cause plants to adapt by altering their chemical composition, their cell structure and how they grow. The set of immediate biochemical and genetic changes is termed the stress- or defense response, depending on whether the inciting agent is an organism or an environmental stress, such as excess light or an air pollutant. Ozone is a common air pollutant that affects plants in urban environments, the productivity of crop plants, and the health of forests at high altitudes. The present project focuses on the response of the model plant Arabidopsis to ozone. Leaf tissues respond very rapidly to ozone gas (even at levels reached in summer in large metropolitan areas) by producing bursts of highly reactive forms of oxygen, called reactive oxygen species. This response is called the "oxidative burst" and serves both a signaling function and as a cell-death trigger. Signals are transmitted within cells by a 3-component (heterotrimeric) G protein, as well as through other signaling pathways, including mitogen activated protein kinase cascades. The present research seeks to understand the role of the G protein in the oxidative stress response to ozone. The first objective is to ask how the components of the heterotrimeric G protein interact with each other, as well as with other proteins, during the ozone-induced stress response, and whether a subunit of the G protein is the direct target of activation in response to ozone. These experiments will be carried out using fluorescence resonance energy transfer, immunochemical methods, tandem affinity purification, and mass spectrometry. The second objective is to identify genes whose transcript abundance is regulated through signals transmitted through the G protein and those regulated through other signals using cDNA microarray expression profiling. Broader Impact: The importance of this work lies in the fact that the metabolic changes that comprise the stress response depress plant productivity; thereby decreasing crop yields. Understanding how plants respond to stress at the molecular level is one of the most important areas for the future of agriculture and sustainable development. Expanding knowledge about molecular and genetic networks activated by stress will open new knowledge-based avenues for enhancing the productivity of plants under suboptimal conditions, a central task in achieving global food security. In addition, this project will also provide training for several undergraduate and graduate students.

Very small 19-25 nucleotide RNAs, termed microRNA (miRNA) and silencing RNA (siRNA), have emerged in recent years as key components of major genetic regulatory mechanisms in eukaryotic organisms. While there are similarities between animals and plants both in the small RNA regulatory mechanisms and in their biogenesis, the details differ markedly. In plants, both miRNAs and siRNAs primarily target complementary RNAs for destruction, while in animals miRNAs largely modulate translation. The 21-22 nt miRNAs of plants are important in meristem function, leaf polarity, floral identity and timing, as well as root and vascular development. The longer 24-25 nt siRNAs are involved in viral and pathogen resistance, transgene and transposon silencing, and heterochromatin formation.

MiRNAs are derived from short stem-loop segments of precursors (pri-miRNAs) encoded in the genome either as separate genes or within the transcripts of conventional genes, sometimes in introns. SiRNAs are derived from longer, largely double-stranded RNA molecules that are either produced endogenously or introduced exogenously. Although originally thought to be separate systems, regulatory mechanisms have been described that involve both miRNA and siRNA. The biogenesis of miRNAs in Arabidopsis involves an RNAseIII-family enzyme called Dicerlike1 (DCL1), a dsRNA-binding protein called Hyponastic Leaves1 (HYL1) and an RNA methyl transferase called HUA enchancer1 (HEN1). It may also require a protein encoded by the recently identified SERRATE (SE) gene.

Previous work in this laboratory established that the HYL1 protein is required for processing pri-miRNA to pre-miRNA. The overall goal of this project is to understand the function of the HYL1 protein in miRNA biogenesis through the following specific 3 objectives: 1) characterizing the HYL1 complex, 2) reconstituting miRNA processing in vitro, and 3) investigating the mechanism of pri-miRNA processing and regulation of miRNA genes. Recent progress in the laboratory has allowed an expansion in possible approaches to identifying the proteins involved in miRNA precursor processing. By co-expressing GFP fusion proteins, it was established that DCL1 and HYL1 co-localize to small perinucleolar bodies that are distinct from the similar Cajal bodies that contain the RNA silencing machinery, designated Microprocessor centers. The recent construction of a number of molecular tools puts the project in a good position to reconstitute miRNA processing in vitro, to rapidly analyze the RNA structural requirements for correct miRNA processing and to define the functions of various proteins in pri-miRNA processing. Finally, the regulation of genes encoding the miRNAs themselves will be investigated.

Intellectual merit. This project will enlarge our understanding of an extremely important aspect of gene regulation involving small RNAs in plants. Studies on the HYL1 protein will enhance our basic understanding of miRNA biogenesis in plants, while the regulatory studies will deepen our understanding of the miRNA regulatory hierarchy.

Broader impact. Viral resistance based on small RNA-mediated mechanisms has already proven valuable in agriculture. Greater understanding of small RNA mechanisms is likely to generate beneficial applications, facilitating the development of inducible small RNA-based mechanisms that may allow plants to withstand more extreme environmental conditions than they can at present without compromising their productivity.