Cell fate determination is a critical process in development of all multicellular organisms. A fundamental feature of sexual reproduction in both plants and animals is the specification of distinct types of cells that give rise to eggs and sperm. So far, little is known about the molecular mechanisms underlying cell fate determination during sexual reproduction in plants. In flowering plants, male reproduction occurs in anthers, which contain highly specialized somatic and reproductive cells. Recently the anther has emerged as a prime model system for the study of cell fate determination and receptor-linked signaling, in addition to its central importance to plant breeding and reproduction. Signal transduction mediated by the EMS1 leucine-rich repeat receptor-like kinase (LRR-RLK) plays a critical role in regulating anther cell differentiation. This project will use molecular genetic, cell biological and biochemical techniques to further analyze the biological functions of EMS1. The expected results would include the identification of an EMS1 ligand and other novel signaling components in the EMS1 signal transduction pathway. The proposed project will shed light on the molecular mechanisms of cell fate determination during sexual reproduction in plants and other organisms. The results may lead to potential agricultural and industrial applications. The proposed research programs will provide excellent opportunities to integrate research into education through training a postdoctoral scientist, underrepresented undergraduate and graduate students, as well as K-12 students.
Intellectual Merit: The world now is facing more severe problems than ever with continuously increasing global demands for food and energy but limited resources on the Earth. Plant sexual reproduction is not only crucial for producing offspring, but also essential for crop quality and yield. The long-term goal of this project is to elucidate how plant sexual reproduction is controlled at the molecular level. In flowering plants, pollen grains (male gametophytes) are produced in anthers. Within each of four lobes (microsporangia) in a mature anther, reproductive cells (microsporocytes/pollen mother cells) are surrounded by four concentrically organized somatic cell layers (epidermis, endothecium, middle layer, and tapetum). After meiosis microsporocytes develop into pollen, while somatic cells, particularly the tapetum, are critical for normal development and release of pollen. So far, little is known about the molecular mechanisms of cell fate determination during anther development. In the excess microsporocytes1 (ems1) mutant in Arabidopsis, the reference species for plant biology, anthers produce excess microsporocytes, but do not form their surrounding tapetal cells. The fact that the EMS1 gene encodes a leucine-rich repeat receptor-like protein kinase (LRR-RLK) indicates that EMS1 mediates signals to control anther cell fate determination. Using integrated molecular genetic, cell biological, and biochemical approaches, the expected outcomes were obtained from two major specific aims that were proposed before. First, the outcomes from this project support that the small protein TPD1 is a ligand of the EMS1 receptor kinase. The results show that TPD1 interacts with EMS1genetically and biochemically. In addition, the ectopic expression of TPD1 causes abnormal differentiation of anther cells. The TPD1 signaling requires a functional EMS1. Furthermore, TPD1 induces EMS1 phosphorylation. Moreover, the secretion of TPD1 is required for the normal function of TPD1-EMS1 signal transduction. Second, genetic and biochemical (yeast two-hybrid) screenings were conducted for identifying novel players in the TPD1-EMS1 signal transduction pathway. More than 4,500 lines have been screened, yielding 10 promising enhancers and repressors. Additionally, three promising candidates were obtained from the yeast two-hybrid screening. This work for the first time shows that the small protein TPD1 serves as a ligand for the LRR-RLK type of receptor kinase EMS1, which provides new insights into the molecular mechanisms of cell fate determination during sexual reproduction in plants and other organisms. Furthermore, identification of novel signaling components will promise a better understanding of the TPD1-EMS1signal transduction pathway in future studies. Broader impacts: The research activities in this project were integrated with science education by promoting teaching, training, and learning. With respect to promoting teaching, two lectures for the Cell Biology and Molecular Genetics courses were created using results from this project. With respect to promoting training and learning, two postdoctoral research associates were trained. Four graduate students participated in this project. Assisted by postdoctoral research associates and graduate students, twenty three undergraduate students were trained through the proposed research activities. Postdoctoral research associates, graduate students, and particularly undergraduate students presented results in conferences. One undergraduate student won the Summer Undergraduate Research Fellowship (SURF) from the American Society of Plant Biologists (ASPB). Two other undergraduate students won research awards from the university. Furthermore, three undergraduate students are co-authors of a research paper. Ten trained undergraduate students pursue their higher degrees in graduate, medical or dental schools. Moreover, a Summer Fun Biology Workshop (SFBW), which contains five labs, was developed and three high school students were trained through this program in two summers. To broaden the participation of underrepresented groups, three undergraduate students (one Hmong American and two first generation college students) were trained. The PI participated in an interdisciplinary conference in the field of regulatory RNAs. To enhance infrastructure for research and education, collaboration were established with two international partners. One international proposal was submitted via collaboration. This project has significant impacts on agriculture, energy, environment, and healthcare. Anther is essential for male reproduction in flowering plants. Male sterility technology is successfully and extensively used by plant breeders to produce hybrid varieties with high yields. Moreover, floral sterility can prevent gene flow from genetically-engineered plants, and increase overall biomass of plants. The TPD1-EMS1 signaling pathway is conserved in flowering plants. Further understanding of TPD1-EMS1 signaling and anther cell differentiation will lead to important agricultural and industrial applications. The collaboration with a scientist at the USDA was established to study the male sterility in sorghum. In addition, anther represents an excellent system to elucidate fundamental mechanisms of cell fate determination. The leucine-rich repeat receptor kinase is associated with Parkinson’s disease. Therefore, this research may benefit healthcare.
