Microscopic valves on the surface of land plants called stomata are essential for plant growth and survival and impact our global environment. Stomata are an interface between plants and the atmosphere. Notably, carbon-dioxide molecules (greenhouse gas) enter plants through stoma and are fixed into sugars to support plant growth and biomass production. Furthermore, a loss of water vapor through stomata, a process known as transpiration, promotes water movement from soil to the tip of plants to support their growth. During plant development, stomata are formed through a series of orchestrated differentiation events, where signals between cells specify the density and distribution of stomata on the plant epidermis. Recent research led to identification of candidate ligands and cell-surface receptors that govern stomatal patterning. This proposal is aiming at establishing the function and action of candidate ligands, EPIDERMAL PATTERNING FACTOR1 (EPF1) and EPF2, and to further determine whether these proteins associate with potential receptors, TOO MANY MOUTHS and ERECTA-family receptor kinases. Accomplishments of the proposed research will advance our knowledge to improve water-use efficiency and biomass production of important crop plant species. The proposed project will provide excellent training opportunities for future scientists at all levels in diverse backgrounds, from undergraduate, a starting technician and a postdoctoral researcher. They will learn broad knowledge of plant biology, molecular genetics, biochemistry, and imaging techniques and how to propose important questions in biology. A part of the research will further be incorporated into the undergraduate teaching lab course (Introductory Biology) to enhance education.
Stomata are microscopic valves on a surface (epidermis) of land plants, including all economically and agriculturally important crop plants grown in the field in the US. Stomatal pores can open and close to enable efficient gas exchange between a plant and the atmosphere. In the adverse environment, such as drought, stomatal pores close tightly to minimize plants from desiccation (wilting). Carbon dioxide acquired by plants through stomatal pores will be fixed into carbohydrates via a process of photosynthesis to produce biomass. Proper numbers and orientation of stomata are critical for growth, productivity and survival of land plants in a changing environment. Moreover, the presence of stomata greatly influences our plant earth. Scientists estimates that a few thousand gigatons of green house gas (carbond dixoide) are fixed into plant biomass (=renewable bioenergy) through stomata in each year; the entire water vapors of our atmosphere are recycled via stomatal pores in every six months. Thus the problems of understanding stomatal development and function have critical implication not only to basic plant science but also to environmental and atmospheric sciences. In order to function properly, stomata require supplies of water and ions from surrounding non-stomatal epidermal cells. Plant cells are encapsulated by cell walls and cannot migrate during development. As such cells becoming stomata (stomatal precursor cells) emit signals that prevent surrounding cells from adopting stomatal cell fate. Molecular nature of genes coding for putative signals (peptide ligands) and receptors have been identified by our group and collaborators. The major aim of this proposal was to demonstrate physical interactions among such signaling ligands and receptors in order to understand the mechanism of how these signaling molecules establish proper stomatal patterns. Ligand-receptor interactions and receptor complex formation were tested using four different systems: 1) Co-immunoprecipitation assays taking advantage of tobacco leaves that transiently produce large amounts of target proteins; 2) Co-immunoprecipitation assays using model plant Arabidopsis expressing functional receptors; (3 and 4) synthetic, biosensor chip platform. Receptors were immobilized on microchips and bioactive, recombinant peptide ligands were applied to the systems via microfluidics. Two techniques, quartz crystal microbalance (QCM)(3) and surface plasmon resonance (SPR)(4) were used to document the binding kinetics of ligands to receptors. Innovation of receptor chip was done as successful collaboration with Materials Science and Engineers at the University of Washington. There are two peptide ligands (EPIDERMAL PATTERNING FACTORS1 [EPF1] and EPF2) that are expressed in the later and earlier stomatal precursors, respectively, and inhibit surrounding cells from becoming stomatal precursors. Our study conclusively demonstrates that EPFs directly bind to the predicted receptors, ERECTA-family receptor kinases, in rapid and sturable kinetics. ERECTA-family receptor kinases formed homo-dimers as well heterodimers with TOO MANY MOUTHS (TMM), a receptor-like molecule without a domain to transduce signals. In contrast, TMM did not associate with itself and exhibited differential binding to EPF1 and EPF2. Furthermore, through specifically blocking receptor signaling, two ligand-receptor pairs, EPF2-ERECTA and EPF1-ERECTA LIKE1 (ERL1), were shown to control two consecutive and critical steps for stomatal development: initiation and spacing. Combined, the work unambiguously establishes the ligand-receptor functions and specificities. In addition to the above work, this grant funding led to the identification of additional, new players of stomatal patterns. The research projects will provide fundamental insight into the molecular mechanisms of cell-cell signaling and tissue patterning, and has a potential to bring critical insight into broad areas of biology beyond plant sciences, including human stem cell and regenerative medicine. Under this NSF funding, a unique, cross-disciplinary collaboration between plant biologists and nanomaterials engineers was established, which led to production of receptor biosensor platform. The receptor immobilization and analysis platform developed in this study can be applicable to test any ligand-receptor interactions in wide-variely of biological systems. In addition, production of bioactive, recombinant EPF peptides from E. coli, opened a door to manipulate stomatal density and hence productivity and drought resistance of important crop plants without any genetic manipulation. This innovation is currently under filing to provisioanl patent application. For educational component, this project provided the training opportunity and career advancement for three postdocs, one graduate student and seven ethnically diverse undergraduates (one Hispanic, four Asian, five female). These trainees presented their work at regional, national, and/or international scientific conferences. Two undergraduates acquired prestigious fellowships (University of Washington Mary Gates Endowment Undergraduate Research Internship, and American Society for Plant Biologists Undergraduate Summer Internship).
