The purpose of the project is to develop and apply methods to sequence translating messenger RNA from specific cell types. An important deficit in current experiments that obtain the list of active genes in plant tissue is that the tissue used comes from a mixture of different cell types, for example, it uses whole flowers or whole seedlings, each of which is made up of many different types of cells. The resulting data on active genes is useful, but is information on the activity of genes from many cells that have different collections of active genes. As cell-type-specific data are necessary to infer gene regulatory networks, current types of data are inadequate to understand a complex organ like a leaf, shoot, or flower. The research will use developing flowers as a system, will adapt existing methods and develop new methods to isolate cell-type specific RNA, and to perform detailed sequencing of the RNA from individual cell types of the developing flower. The result will be a set of tested methods that allow researchers to isolate messenger RNA from single cell types found in complex tissues, and therefore to record patterns and changing patterns of gene activity from each of the single cell types found in developing organs.
Broader Impacts The broader impacts of the project are in three areas. First, the completion of the proposed research will result in new technologies for the entire plant science community to study RNA and gene activity at cellular resolution in virtually any cell type, which is expected to be crucial for the understanding of various questions from development to energy capture and response to the environment. All the methods and lines generated through this project, which will enable cell-type specific analysis in other laboratories, including those of young and starting researchers who would have to spend considerable time to make such lines, will be made freely available to the research community. Second, by being partly established within the environment of a much larger group of undergraduate students, graduate students, postdoctoral fellows and technical staff that are engaged in different research projects on Arabidopsis and animal development (the Meyerowitz laboratory and the personnel of the Caltech Jacobs Center for Genetics and Genomics), the project will provide familiarity with a variety of genomic technologies and approaches to a substantial pool of future independent investigators. This pool will include undergraduate summer researchers and students from underrepresented minorities recruited through established programs at Caltech. Finally, public outreach and training activities will include provision of the materials generated (especially images) for the "Grounding in Botany" program, a collaboration between the Meyerowitz lab and the Huntington Botanical Gardens, in which laboratory exercises using plants are developed by teachers for high school classrooms; and for a program in which graduate students and postdoctoral scholars from Caltech spend up to six weeks at the Exploratorium in San Francisco, teaching high school and elementary science teachers in a summer teacher training program.
New methods of genome-wide analysis are permitting plant scientists an unprecedented view of the dynamic processes that occur during the development and growth of plants. Particularly revealing is transcriptome analysis, by which the population of messenger RNAs present in a plant at any time or after any treatment is determined, as it allows a listing of the genes active or activated at different times in development, or after different treatments, and therefore provides a quantitative view of plant processes and plant responses. Next-generation nucleic acid sequencing has greatly potentiated this approach, as it is both more sensitive and more accurately quantitative than previous approaches. There is a fundamental problem, though, with the way that next-generation sequencing methods have been applied to transcriptome analysis in plants and in animals. So far, these methods have been used on tissues, or on whole plants, so that the transcriptomes of many different cell types are mixed, thus confusing the analysis and interpretation of the data. In this project we developed a method for the isolation of RNA from single cell types, as defined by the activity of any chosen gene promoter, and then using next-generation sequencing methods to determine the levels of each RNA type isolated. The method, called TRAP-seq, was an adaptation of existing methods for immunological purification of polyribosomes based on introducing to a plant an immunologically tagged ribosomal protein whose expression is coupled to activation of a cell-type specific promoter, or DNA regulatory region. The ribosomal protein is thus only expressed in a single cell type, and immunoprecipitation of ribosomes from the whole plant recovers only those from the single cell type, along with the RNAs they are in the process of translating to proteins. Subsequent next-generation sequencing of a DNA library made from the precipitated polyribosomes allows a readout of all of the RNAs being translated in a single cell type – therefore giving a cell-type specific resolution to transcriptome analysis. In addition to demonstrating that the immunoprecipitation method works as a method for obtaining samples for next generation sequencing, we have published and made available in public databases the full transcriptome analysis of specific cell types in three domains of developing flowers, the cell types characterized by the promoters of the genes APETALA1, which acts in specification of sepals and petals, APETALA3, which specifies petals and stamens, and AGAMOUS, responsible for formation of stamens and carpels. A generally expressed promoter was also used to provide cell-type nonspecific controls. In the course of these studies we discovered a class of ribosome-associated noncoding RNAs, which indicate as-yet unknown processes of gene regulatory control that are under study. We have subsequently produced six additional cell-type specific gene constructs, using six additional promoters active in single domains in developing flowers and shoot meristems. The broader impact of this work is at multiple levels. Not only has a new and generally useful method been reduced to practice and made generally available for use in plant and animal studies, and developmental transcriptome analysis for multiple floral domains been made available, but 10 gene constructs have been produced and tested, and made available for the use of other laboratories. The published paper has been cited by other labs, including in a paper entitled "Arabidopsis paves the way: genomic and network analyses in crops," indicating that the methods used in Arabidopsis are expected to be useful in agriculture. And, the personnel engaged in the project have used their records of accomplishment to develop their careers by establishing their own laboratories.
