David Jackson, Ph.D. - US grants

This award supports the acquisition of a scanning electron microscope (SEM) that operates under conventional high vacuum as well as variable pressure mode. The instrument will by used by at least seven research groups at the Cold Spring Harbor Laboratory to study the developmental genetics of plants and animals. Projects that will use the microscope include the analysis of leaf and meristem development in maize and Arabidopsis; analysis of the Arabidopsis flower and ovule development; the developmental genetics of cell death in the nematode C. elegans; developmental biology of GTPase signaling in Drosophila; and the analysis of morphological changes associated with the development of learning and memory in the Drosophila brain. The SEM is essential for these studies.
The Plant Group at Cold Spring Harbor Laboratory is engaged in a systematic search for new mutations in Arabidopsis using gene trap transposon mutagenesis. The SEM will provide a means to rapidly characterize these mutations in terms of morphological changes as well as changes in cell shape or specification. In addition the SEM will be used for training graduate students and postdoctoral fellows, and in the Cold Spring Harbor Laboratory course in Arabidopsis molecular genetics, which has a wide impact on the plant field, and in the DNA Learning Center at CSHL. The ease of use of the variable pressure SEM, compared to conventional SEMs, allows samples to be visualized within minutes of dissection without the need for extensive sample preparation, and the automated settings and the standard operations in 'Microsoft Windows' format means that users can be quickly trained. Therefore the impact of this microscope on research and education at Cold Spring Harbor Laboratory will be substantial.

The goal of this project is to explore the use of high-throughput proteomics to determine gene function based on the cellular interaction network of an encoded protein. It is appropriate for a Small Grant for Exploratory Research because it "ventures into emerging and potentially transformative research ideas". Specifically, methods will be developed by which the protein: protein interactions of rice genes of unknown function can be determined. This information will allow identification which protein(s) with known cellular role(s) interact with a given unknown protein, and will therefore will link the unknown protein to that cellular function. The information will be captured in a publicly available database. The data will also allow construction of broadly based protein interaction maps of rice.

Graduate raining will be provided in areas that will combine genome sequence analysis with protein interaction analysis. Such skills will be crucial as the community grapples with integrating these disparate, large datasets. In addition, the approached developed could create a paradigm shift that could have a significant impact in defining a new strategy for analyzing complex plant genomes.

The data resulting from the work will be available at:
http://www.cshl.edu/genseq/riceproteome.

Maize cells consist of interconnected but discrete compartments that help to maintain cellular function and order. Identifying proteins that localize to these compartments is critical to understanding developmental and physiological processes in maize, which in turn provides guiding information for crop improvement. This project makes use of recent advances in genomics to identify proteins that localize to diverse cellular compartments. The project will generate reporter lines that express proteins tagged with a fluorescent marker. Using confocal microscopy, the lines will display visual information about when and where the tagged proteins are expressed and how specific proteins may be interacting and functioning. Genes for tagging will provide complete marker coverage of cellular compartments. Several resources will be used for gene selection, including known genes from the auxin/cytokinin hormone pathway and from the RAB-mediated vesicle trafficking pathways. Gene selections will also be guided by public gene models generated from maize genome sequencing projects and by specific requests from the maize research community. The outcomes of the project will include a set of stable tagged lines expressing fluorescent protein-derived tags for 100 proteins, which will represent full coverage of cellular compartments. The tagged gene constructs will be freely available from the Jackson lab, and seeds of transgenic lines will be available from the Maize Genetics Stock Center.

Access to project outcomes:
Data on characterization of the lines at the cellular level will be compiled in a localization catalog posted on a public website at http://maize.tigr.org/cellgenomics. Cell biology workshops will help train the scientific user community in analysis of the tagged lines using confocal microscopy. The project will also serve tribal community colleges in the Rocky Mountain West by enhancing education in cell and molecular biology. The project will therefore generate resources that bridge cell biology and functional genomics and will provide training across diverse scientific and learning communities. Training and outreach activities will be reported on the Plant Genome Research Outreach Portal at http://www.plantgdb.org/pgrop/pgrop.php.

Jackson, David. NSF 0642707. Hormonal and Genetic Networks in Phyllotaxy.

Plant development proceeds through the continuous initiation of organs from groups of stem cells called meristems, and shoot organs such as leaves and flowers, are initiated in regular patterns known as phyllotaxies. The aim of this project is to understand how these patterns are generated, a question that has been of long-term interest to mathematicians, physicists and biologists.
In this project, genes that regulate leaf initation and phyllotaxy will be studied. Mutations in these genes cause plants to make extra leaves, and develop with opposite phyllotaxy rather than the alternating pattern that is normal for maize. One of the genes, ABPHYL1, has been isolated, and encodes a cytokinin response regulator homolog. Cytokinin is a plant hormone, and the regulation of ABPHYL1 and its interaction with another important plant hormone, auxin, will be studied. Genes that are regulated by ABPHYL1 have also been identified, and their functions will be elucidated. A second locus, ABPHYL2, also regulates phyllotaxy, and this gene will be isolated. The impact of this work will be to provide a greater understanding of the cross talk between plant developmental and hormone signaling. The studies could also lead to crop improvement, since the initiation of leaves in regular patterns optimizes light capture for efficient photosynthesis, and in some plants provides architectural support.
These broader impacts of these studies will be through contributions to several areas, including developmental regulation, morphogenesis, and hormonal signaling. The studies will also be of interest to researchers in other disciplines, such as mathematics and computer science. In addition, a postdoctoral fellow, graduate student and undergraduate or high school students will be trained in plant developmental genetics and bioinformatics. Materials generated from the study will be used in teaching at CSHL and at the Dolan DNA Learning Center.

The Cereal Genomics Workshop at Cold Spring Harbor Laboratory will give plant biologists the skills necessary to navigate the increasingly complex genomics information landscape, and to identify and encourage young researchers who might choose plant genomics as a career by exposing them to the field's practice and practitioners. A major aim will therefore be to enable students and post docs to take advantage of emerging genomics data in the cereals. Specific problems and unique challenges exist in cereal genomics. Many of the genomes (for example, maize, wheat, barley) are large and complex, and it is unlikely that we will have completely mapped and sequenced genomes for these species in the foreseeable future. The cereals also provide the primary source of nourishment to the world's human and domesticated animal populations. This workshop will provide young students and scientists with the tools and skills they need to exploit the emerging genome data in all cereal crops including complete genome sequences (rice, maize, sorghum) and extensive EST and genome survey sequence from wheat and other grasses.

The broader impacts of this workshop will be in exposing young students to the excitement of cereal genomics, as well as enabling more senior researchers to develop cutting edge research and curricular activities. It will also foster development of collaborations and cooperation among the participants.

The REU Site at Cold Spring Harbor Laboratory (CSHL) will provide a research program for undergraduates during the summers of 2009-2011, through support provided by the Directorates for Biological Sciences (BIO), Mathematical and Physical Sciences (MPS), and Computer and Information Science and Engineering (CISE). Six students will be selected to participate in a 10-week research program in bioinformatics. The REU program is part of a larger summer Undergraduate Research Program (URP) at CSHL, providing an opportunity for students to learn about bioinformatics and computational biology by conducting intensive research under the supervision of a mentor. Students learn about scientific reasoning, laboratory methods, theoretical principles, and scientific communication. In addition to research, participants attend seminars, workshops, panel discussions on careers in science, lectures on ethics, and special events designed specifically for them. At the URP Symposium held at the end of each summer, students present a summary of their research to their peers and advisors. Students also write a final report, similar to a scientific abstract, to describe his or her project. Students are also encouraged to attend advanced courses at CSHL to learn the latest experimental techniques in different fields and to seek advice about their future schooling and research. By the end of the program, students are well equipped to decide whether scientific research is a career they would like to pursue. The program is open to all US citizens or permanent residents, and applications from under-represented minority groups are encouraged. Further information can be obtained by visiting www.cshl.edu/URP/nsf-reu, or contacting the Watson School of Biological Sciences at 516-367-6911 or urpadmin@cshl.edu, or the Program Director, Dr. David Jackson, at jacksond@cshl.org (tel 516-367-8467).

Funds are requested to support attendance at the 7th International Conference on Plasmodesmata, which will take place from 21st-26th March 2010, in Sydney, Australia. Plasmodesmata are tiny channels that connect plant cells to their neighbors, and are important for plant growth and crop yields because they control the movement of nutrients and spread of plant diseases. Because research in this field is multidisciplinary, and extremely active, it is important that researchers in diverse subject areas have the opportunity to meet and discuss their research. Many important discoveries have been made in this area by US scientists. Plasmodesmata research is also highly interactive, and has many international collaborators who are essential to advance the science rapidly. Therefore attendance at this conference by a diverse pool of US scientists will be highly beneficial both to basic research and to agriculture.

It is expected that the conference will contribute significantly to science and education. First, the conference will foster interactions between scientists with skills in diverse areas. Second, involvement of students and post docs will be strongly encouraged by the secluded and interactive environment, and by having each participant present a talk or a short oral presentation alongside his or her poster. Participation by minority and female scientists will also be strongly encouraged. This will be achieved by identification and encouragement of individuals, as well as by advertising the conference widely. Success of the conference will be assessed by a written questionnaire distributed among all attendees.

Abstract

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Funds are provided for Cold Spring Harbor Laboratory (CSHL) to renovate and upgrade existing plant growth facilities at our Uplands Farm Research Field Station. CSHL has an extensive greenhouse complex offsite where seminal work in Arabidopsis and crop plants has been conducted. The research focus on crop yield and adaptation of crops to substandard conditions has global impact and this will increase with these improved facilities. The greenhouses and growth rooms are used to support the research programs and also support the plant genetics teaching programs of the CSHL Dolan DNA Learning Center. These aging and outdated facilities are inadequate to meet the demands of current genome driven plant biology research. The infrastructural improvements will provide appropriate growing conditions for a greater diversity of plant species and will increase the energy efficiency of the facilities. In the proposed renovations, CSHL intends to:
1 Replace boilers in two smaller greenhouses, and two boiler burner units in the largest of the three greenhouses.
2 Install updated evaporative cooling units in each of the three greenhouses.
3 Install automated ventilation, sunshade, and irrigation systems in all three greenhouses.
4 Replace the aging, hazed acrylic sheathing on the largest greenhouse.
5 Renovate and improve the head house of the largest greenhouse.
6 Replace the cooling unit in the Field Station Laboratory with a modern and more efficient unit with an economizer ventilation unit.
7 Replace outdated lighting fixtures in the Arabidopsis growth facilities with improved, high-efficiency units and install a dedicated heating loop.

Living organisms are made up of smaller units called cells, and these cells need to communicate with each other to coordinate how they grow and develop. Plants have evolved a way to communicate by making tiny channels to connect their cells. Many different types of molecules pass through these channels to feed growing tissues and to instruct the plant how to grow. This project aims to understand how the passage of signals through the channels is controlled, by studying a newly discovered factor that controls this process. It will also screen for new factors that influence the way in which the channels instruct the growth of special types of cells called stem cells that are essential for plant growth. These studies have the potential to make significant improvements to agricultural productivity, and to limit the spread of plant diseases, which sometimes use the channels to spread through the plant. In addition to the scientific and technological advances detailed above, this project will train young scientists at various levels, as well as developing resources to involve high school students in cutting edge biology research. The PI directs the Partners For the Future Program at CSHL, which immerses local high school students in active research at Cold Spring Harbor Laboratory. He will also develop an educational exchange with an all-female minority serving high school in Brooklyn, New York. This activity will expose the excitement and applications of molecular biology to students who otherwise have little exposure to scientific research.

Plasmodesmata are microscopic channels that connect plant cells to integrate growth, development and nutrient availability, providing organism wide connectivity. Plant development relies on pluripotent stem cells in specialized niches called meristems, and the movement, or "trafficking", of homeodomain transcription factors through plasmodesmata is required to maintain the stem cells. This research will develop and adapt state of the art methods in protein-RNA interactions, RNA localization and proteomic analyses to study a newly identified RNA binding protein that is required for trafficking of stem cell regulatory proteins. It will also test the hypothesis that the newly identified protein interacts in a protein-mRNA complex to facilitate passage of transcription factors through the plasmodesmata. In an independent approach, the research will also use a proteomic screen to identify new factors that control PD trafficking. Movement of protein and RNA signals in plants is critical for their development, as well as how plants respond to the environment. The results of this project could therefore allow the manipulation of plants to improve agricultural productivity. The project will also integrate training of junior scientists, including minority high school students, in molecular genetics research.

This award was co-funded by the Physiological Mechanisms and Biomechanics Program in the Division of Integrative Organismal Systems and the Cellular Dynamics and Function Cluster in the Division of Molecular and Cellular Biosciences.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

PI: David Jackson (Cold Spring Harbor Laboratory)

Co-PIs: Anne Sylvester (University of Wyoming)
Agnes Chan (The J. Craig Venter Institute)

Intellectual Merit. Maize is a powerful model system for functional genomics in the grain crops, because its reference genome has recently been sequenced and functional tools are increasing. However, new methods of investigating gene function within cells and tissues are needed to understand protein functions at the cellular and sub-cellular level, which will ultimately provide guiding information for crop improvement. This project aims to generate tools in maize that can be used to drive expression of any gene in a number of specific chosen tissue or cell types. The use of these tools will permit the visualization of reporter genes with unprecedented spatial and temporal resolution, and will also enable functional gene over-expression or gene knockout studies. In addition, this project will make use of newly improved fluorescent protein color tags to label sub-cellular or developmental compartments, and will develop live imaging techniques for growing maize plants to demonstrate the utility of all of the reporter lines for functional studies. The project will therefore transform functional genetic experiments in maize, by providing a means to understand gene function in specific cells and tissues during development. The project will deliver to the research community a permanent stock of stably transformed seeds for 50 tissue specific activator/reporter lines, 20 fluorescent-tagged protein lines, as well as fully characterized in vivo imaging methods. It will also develop a pipeline for handling large image datasets that will be applicable to other projects. All resources, including images, seeds and gene constructs, will be publicly available via the project website http://maize.jcvi.org/cellgenomics. For long-term availability, images will be migrated to the community database, MaizeGDB, and seeds will be deposited with the Maize Genetics Stock Center.

Broader Impacts. The project will integrate education and outreach by organizing an annual Genetics Workshop for Native American students at Little Big Horn College, a Tribal College to the Crow Nation. The project will also integrate functional research into educational tools by generating 3D interactive animations of a maize cell and growing plant, displaying sub-cellular compartments and gene expression data. The animations will be beta-tested by three target groups, including Native American students at Little Big Horn College, deaf and hard of hearing students in a cell biology class at Gallaudet University, and University of Wyoming biology students. Animations will be made publicly available and incorporated in course curricula taught at the participating institutions.

Plant genome research over the past 20 years has provided a deep understanding of genetic pathways that underlie economically important processes in crop plants. However, as in most organisms, many plant genes have "backup" copies, or duplicates representing genetic redundancy. Very little is known about the effect of such redundancy on plant improvement efforts. This lack of knowledge complicates the efficient use of genetic resources. This project will focus on a known group of signaling genes to understand the basic principles that underlie genetic redundancy in plants. It will therefore advance knowledge in a fundamental area of plant genome biology. Outcomes from this project will have the potential to bring improvements to US agriculture by providing new knowledge and tools to develop high yielding crops. The project will also train a number of young scientists at various levels, as well as promote outreach and education in plant genomics. Project personnel will develop new teaching modules to highlight the importance of plant genomics in crop domestication, and will present these in schools and workshops that target female students and underrepresented minorities. Outreach activities will also target rural farming communities in the New York, Massachusetts and North Carolina areas, where open house displays and lab visits will be used to educate these groups about the importance of plant genomics research in agriculture.

This project asks how redundancy in signaling pathways has evolved across the plant kingdom. It will develop a genome-level understanding to link genes and pathways to complex phenotypes, by testing the hypothesis that genetic redundancy in plants is controlled by Responsive Backup Circuits (RBCs). A second hypothesis to be tested is that signaling network outputs can be modulated and exploited using weak promoter alleles. Three species will be used, the model system Arabidopsis, to rapidly test hypotheses, and tomato and maize, divergent and economically important crop species. Genetic redundancy is a major limitation to the ability to link genes to phenotypes in plants, and this project will use a subset of Leucine Rich Repeat Receptor Like Kinases and their predicted ligands as a model network. Signaling genes selected by phylogenetic analysis will be targeted for knockouts using genome editing technologies (CRISPR/Cas9). Genome-wide transcript profiling will then be used to deduce redundancy mechanisms and reiteratively design new knockouts to address the effect of disrupting redundant paralogs. At each stage, careful phenotyping will be used to understand the effect of multiple gene knockouts at different developmental stages relevant to crop productivity. Redundancy in gene regulatory sequences (promoters) will also be addressed by developing a generalizable CRISPR/Cas9 multiplex knockout strategy to make semi-random mutations across gene regulatory sequence regions. These lines will be screened en masse, and represent a new approach to mutagenesis in plants, with a potential to generate new genetic diversity, and to recover weak alleles with enhanced yield traits.

PI: Thomas Gingeras (Cold Spring Harbor Laboratory)

CoPIs: David Jackson, Robert Martienssen, W. Richard McCombie, Michael Schatz, and David Micklos (Cold Spring Harbor Laboratory), Doreen Ware (USDA-ARS/Cold Spring Harbor Laboratory); and Ken Birnbaum (New York University)

Maize (corn) is one of the most economically and agriculturally important crops grown in the world. It has assumed this position after centuries of careful genetic breeding to enhance many of its growth and nutritional properties. The generation of high quality genome sequences paired with diverse molecular data allows scientists to better understand the effects of this selective breeding at both the genetic and epigenetic levels. The MaizeCODE project aims to create a comprehensive reference encyclopedia of highly useful genomic reference sequence resources for breeders and plant scientists to use to improve economically important crop traits like disease resistance and yield. In addition, this project will provide broad and comprehensive training opportunities for students, breeders and practicing scientists through specific courses and workshops that will address various approaches to obtain and analyze MaizeCODE data. The Education, Outreach, and Training (EOT) effort will prepare faculty from primarily undergraduate institutions (PUIs) to analyze MaizeCODE with undergraduate students and will provide travel awards for graduate students to attend MaizeCODE training at professional meetings. The EOT program will be unique in promoting Science, Technology, Engineering and Math (STEM) disciplines by anticipating and encouraging broad participation in primary data analysis by undergraduate and graduate students.

Improved assemblies of the maize genome will provide a foundation for the identification of biochemically active and biologically functional elements encoded in this working-draft sequence. A comprehensive catalog of these elements will be a critical component in strategies to link genotype with important traits in maize, a classical genetic system. This is the overarching goal of the MaizeCODE project. The human ENCODE project is a model for such a comprehensive catalog. Building on the project team's leadership experience with ENCODE, this similarly integrated and multi-disciplinary project has three main objectives: 1) to develop high-quality working drafts for two inbred maze lines and one teosinte inbred line, 2) to identify regions of the maize genome that are transcribed, methylated, bound by specific modified histones in six cell types that are the major progenitors of the root and shoot systems (focusing on histone modification in three root cell types) and transcription factors in five unrelated tissues and 3) to store, collate, display and disseminate the data to the broader community of plant biologists worldwide. Given the wealth of "genome to phenome" studies in maize, and the emerging realization that much of the variation under selection acts at the level of gene regulation, it is expected that this project will have broad and significant impact on maize genetics research and breeding, with the potential to inform similar research in other grass crops. The project expects to extend significantly the functional annotations and the current understanding of the regulation of gene activity in maize, adding critical content to established databases and graphical genome display centers. Information generated in this project will be rapidly and broadly disseminated using publically available databases and the CyVerse (www.cyverse.org) online resource.

Crop plants provide food, feed for livestock, and other essential materials. Breeders are continuously improving our crops; however, there is an urgent need to accelerate crop improvement in the face of climate change and limited resources. Natural genetic variation in the form of DNA mutations is widespread in crops, and is the starting material for their improvement, but such variation is often not useful or is unpredictable in its effect on plant growth. Genes that control important yield traits are expressed at specific levels, locations and times during plant growth, and tuning these expression programs may enhance crop productivity. Gene expression is controlled by regions of DNA surrounding genes known as cis-regulatory elements. Despite their fundamental biological significance, the identification of such elements and their use in agriculture has been challenging. This research project, a collaborative effort between scientists at Cold Spring Harbor Laboratory, the University of Massachusetts-Amherst, and the Hebrew University of Jerusalem, will predict regulatory elements using a newly developed computational algorithm, Conservatory, combined with existing genome sequences from many plant families. These elements will then be modified using CRISPR genome editing tools. These new variants will be tested for changes in phenotype that lead to improvements in yield and other important agronomic traits. The project will train young scientists at various levels, as well as promote outreach and education in plant genomics in partnership with Genspace, a Community Biology lab in Brooklyn, NY. The project will develop a new curriculum for high school students from under-resourced Title I schools and demographic groups historically excluded from the life sciences to explore applications of CRISPR in agriculture, including hands-on labs in plant transformation and CRISPR editing.

This project will test the hypothesis that genes with conserved functions are regulated by deeply conserved cis-regulatory elements (CREs) across angiosperms, and that characterizing these CREs will provide a new level of understanding in linking genotype to phenotype. The project will exploit the recent explosion in high-quality sequenced genomes to identify conserved regulatory elements across angiosperm diversity using the Conservatory algorithm. The functions of the elements identified by Conservatory will be tested by precise genome editing, with a focus on developmental regulators and architectural traits. Functional dissections will be performed in two species in each of three diverse plant families, spanning eudicots and monocots, which will allow the assessment of CRE functional evolution over shallow and deep timescales. The catalog of conserved regulatory elements identified, and the editing strategies developed to test their functions, will reveal fundamental principles governing gene expression control and will accelerate innovative approaches to fine-tune crop productivity traits. Critically, the tools, techniques and fundamental principles emerging from this multi-disciplinary project will comprise a valuable community resource, enabling the engineering of diverse systems and phenotypes, such as biotic and abiotic stress tolerance, nutritional quality, and symbiosis. All project outcomes will be widely accessible through long-term public data and genetic repositories.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.