CoPI: W. Zacheus Cande (University of California, Berkeley; subawardee)
Senior Personnel: Lisa Harper (University of California, Berkeley/USDA-ARS)
As complex organisms develop, the body plan is established first, followed by the specification of organs, then their constituent tissues, and finally the functional cell types are designated. The regulation of cell fate acquisition occurs at many levels and is reflected in the gene expression and protein content within the cells and includes responses to external information such as hormones provided by surrounding tissues. Despite the importance of cell fate to final function, this process is difficult to study in complex tissues with many cell types and because the process is occurring asynchronously in many tissues. This project will exploit the five major cell types and their synchronous development within anthers to dissect cell fate in maize. Gene expression and protein changes that distinguish cells from their neighbors with a different fate will be defined. Mutants defective in cell fate acquisition at specific stages of anther development will be recovered by screening large collections of male-sterile mutants of maize established by prior work. The normal progression of cell fate acquisition will be elucidated by comparing cell expression patterns of normal to mutant anthers. Selected mutants will be described cytologically and by their pattern of gene expression. Finally, genes defining key mutant stages will be cloned to determine the nature of the gene product essential for normal cell fate acquisition. Data generated in the course of this project including microarray data and cytological descriptions of mutants will be made available through GEO (Gene Expression Omnibus (GEO) (www.ncbi.nlm.nih.gov/geo/) and through MaizeGDB (www.maizegdb.org). Biological resources will be available through the project and through the Maize Genetics Cooperation - Stock Center (http://maizecoop.cropsci.uiuc.edu).
The lack of synchrony in pre-meiotic cells in most plants and animals has precluded deep analysis of the steps required for this particular fate decision. Consequently, new knowledge and insights from this project will illuminate steps in cell fate specification for the meiotic cells and surrounding somatic cells that will be of general significance in understanding both complex plant and animal development. To acquaint undergraduate students of agriculture focus with modern methods in plant genetics, the gene-tagging component of the project will be conducted at CalPoly-San Luis Obispo. Project senior personnel will acquaint students with the theory of transposon tagging in plants, and then train students to screen for male-sterile mutants in the field, to conduct genetic crosses for propagating mutants, to conduct allelism tests with previously identified mutants conferring similar phenotypes, to dissect anthers for analysis of gene expression, and to prepare DNA samples for the cloning of newly tagged mutant alleles.
Normal 0 false false false false EN-US X-NONE X-NONE Control of pollen production in crop plants such as maize is fundamental to producing hybrid seed commercially and a fascinating problem in genetic control of development. Within flowers, stamen primordia are initiated and contain a few hundred cells; within 10 days, there are 50,000 cells in a four lobed anther plus a filament that connects the anther to the main plant body. Within each lobe, about 150 cells are specified for meiosis, ultimately producing pollen containing the sperm required for sexual reproduction. All other anther cells play supporting roles in maintaining meiotic cells and the pollen. Because plants lack a germ line of cells dedicated for reproduction (as found in animals), specification of meiotic cells from of somatic cells has been a great botanical mystery. This project classified hundreds of maize male-sterile mutants as pre-meiotic during the first 10 days of anther development, during the 6 days of meiosis, or during the two weeks after meiosis when pollen matures. Supplementing the historic collection of pre-meiotic mutants an additional 23 loci were defined. These were ordered by time of action and described histologically to pinpoint which cell type fails. To gain insight into processes regulated these genes critical for normal development, we proposed cloning three of historic mutants -- this was accomplished early in the project (ameiotic1, mac1, ms8) and a fourth mutant was published independently (msca1). Project personnel also cloned ms23 and ms32, and cloning is in progress or being verified for mtm00-06, tcl1, ms*6015, EMS63089, and ms9. For these mutants, confocal microscopy was conducted to pinpoint developmental failure more precisely. The mutant analysis is conducted against a detailed description of normal maize anther development, a landmark accomplishment relating anther size to internal developmental events including cell dimensions, cell division patterns, and cell numbers in each tissue throughout anther ontogeny. Complementing the microscopy, gene expression patterns of many of mutants were compared to normal fertile siblings before and after developmental failures. These studies pinpointed key processes that must occur in a specific temporal order to achieve normal cellular function. By analyzing mutants with similar defects common themes were identified, and the impact of individual mutants refined. The proteins typical of normal and male-sterile mutants were also analyzed using a deep proteomics (high throughput protein identification) methodology to address whether genes expressed as RNA produce the expected protein product, and at what stage these proteins are present. Experimentally, we discovered how maize meiotic cells arise. This key step is regulated by the MSCA1 protein, which acts as a switch to sense oxygen levels. At low levels, the MSCA1 protein triggers meiotic specification measured as very rapid cell expansion and secretion of the MAC1 protein. MAC1 instructs neighboring cells to divide in a particular way to establish the somatic helper cells that nurture the meiotic cells throughout anther development. These events occur on days 1 and 2 of maize anther development, much earlier than previously hypothesized. The discovery that low oxygen triggers meiotic cell specification provides a mechanism that relies on intrinsic growth properties and environmental conditions; the control mechanism can be readily manipulated by altering the level of oxygen surrounding the developing anthers, resulting in the discovery than any anther cell has the potential to become a meiotic cell -- there is no special position within the anther, instead all cells can respond to low oxygen. The regularity of normal development is achieved by the anatomy of the anther lobes. Broader Impacts: An unexpected project discovery was identifying a highly anther-enriched class of small RNAs (called phased small RNAs that are either 21 or 24 nucleotides long); these "phasiRNAs" are only found in grass flowers such as maize and rice. We found that a specific 22 nucleotide "trigger" RNA is required to produce each length of phasiRNA; the trigger molecule for the 24 nucleotide phasiRNA class is also unique to grasses. Mutants with abnormal meiotic cells (msca1 and mac1) fail to make the 24 nucleotide phasiRNA and the required trigger molecule. In the ocl4 mutant, the meiotic cells are normal but supporting somatic cells are aberrant, causing male-sterility; here, the 24 phasiRNAs are more or less normal, but the 21 nucleotide phasiRNAs and the corresponding trigger molecules are missing. Discovery of these grass anther phasiRNAs has opened up an entirely new line of investigation. Educational activities for agriculture focused-students from Cal Poly-San Luis Obispo was a major training activity. These students ran summer genetic screens at Cal Poly with the Principal Investigator and learned molecular genetic techniques at Stanford. Annually ~10 Stanford and UC-Berkeley undergraduates participated in the project, three Ph.D. students, and 6 postdoctoral scholars conducted multiyear studies as part of the project, with several additional shorter term postdoctoral scholars participating peripherally or just now starting on the project.
