During development, all multicellular organisms face a similar set of challenges: they must create a diverse set of specialized cell types, organize these cells into functional tissues, and maintain pools of stem cells to replenish those tissues throughout their lifetimes. Asymmetric cell division, in which one cell divides to create daughter cells that differ in size, location, and cellular components, has long been recognized as a mechanism important for cell fate. More recently, there has also been tremendous interest in how asymmetric cell divisions create and maintain stem cells.
My lab uses the development of plant stomata (the epidermal structures that regulate CO2 and water vapor exchange between the plant and atmosphere) as a model for the study of cell fate, stem cell self-renewal, and cell polarity in plants. The stomatal lineage distills many of the features common to all tissue development: stomatal precursor cells are chosen from an initially equivalent field, they undergo asymmetric and self-renewing divisions, they communicate among themselves, and they must respond to information from a distance. We believe that the study of plant stomata can reveal novel mechanisms of asymmetric cell division and new solutions to the problem of balancing the robust specification of cell types with the ability to alter development in the face of injury or environmental change.
Stomata provide a framework to study the fundamental processes of plants at different organizational levels, from molecules and cells to whole plants and ecosystems. Stomatal development can impact human health and well-being at two distinct levels: (1) as a model for self-renewal that can inform analogous processes in humans, and (2) because of the function of stomata in photosynthesis and plant-atmosphere interactions. Stomata are essential for photosynthesis in all large terrestrial plants; at a fundamental level, human health and well-being are inextricably tied to the activities of plants in capturing sunlight energy and producing material that serves as our food, fibers, and fuel.
Generating Predictive Models of Stem Cell Behavior
After pioneering the use of transcriptional profiling to identify stomatal regulators, my group was the first to identify a gene required for stomatal development. We also identified three "master regulator" basic helix-loop-helix (bHLH) transcription factors that sequentially control stomatal development and connect this process to the molecules and logic that regulate muscle and neural development. Our research has shown that this basic transcription factor network operates (with interesting modifications) in diverse plants, including food crops and native grassland species.
Focused experiments on individual bHLHs allowed us to connect the broadly used mitogen-activated protein kinase (MAPK) signaling pathway to the initiation of stomatal stem cell divisions via its direct phosphoregulation of one bHLH. We recently extended this specificity theme by demonstrating that other bHLHs recruit core cell-cycle regulators and homologs of human tumor suppressors to ensure cells stay stably committed to stomatal cell fates. Because these master regulator bHLHs are expressed in discrete epidermal cell types, we created a "stomatal toolkit," using enhancers from these genes to activate or eliminate gene expression in specific cell types. Expressing fluorescent proteins with these enhancers enabled us to FACS-sort pure populations of individual cell types. These technical capabilities allowed us to identify unexpected behaviors among MAPK family members. It is now possible to move our developmental questions to a larger scale: What are the whole-genome transcriptional, translational, and epigenetic changes that take place as cells transit through phases of acquiring, maintaining, and leaving self-renewing fates?
Creating Cell Polarity and Asymmetric Divisions in Plant Cells
Once the decision is made to undergo a fate transformation—to enter the stomatal lineage or to self-renew—how do plant cells generate the necessary asymmetric and oriented cell divisions? Asymmetric divisions are universally used to create cell-type diversity and pattern, but structural constraints imposed by plant cell walls, combined with the absence of recognizable homologs of animal or fungal cell polarity genes, require plants to utilize new molecules and mechanisms to create cellular asymmetries. Therefore, when faced with the same asymmetry generation problems, plants and animals have arrived at different solutions. If we are to understand how their functional cell types are made, we must study plants directly. The different solutions may also be useful as "work-arounds" in animal cells to bypass defects in the endogenous system or to adaptively alter the system's behavior in a bioengineering context.
Identification of the First Plant "Polarity Protein" and Development of a Polarity Module
Our first breakthrough in the elusive plant polarity program was BASL, a gene required to generate the physical and fate asymmetry of stomatal-lineage cells. We showed that BASL protein is asymmetrically localized at the cell periphery prior to asymmetric divisions and that only one daughter inherits this peripheral BASL. BASL can also localize to the nucleus, and monitoring localization over time revealed that BASL's subcellular localization is predictive of cell fate. BASL demonstrates that plants are able to localize and segregate proteins during asymmetric divisions. Moreover, when ectopically expressed, BASL-GFP (green fluorescent protein) localizes polarly in additional cells. All plant cells, therefore, must have some mechanism for localizing and maintaining polar BASL. Previous studies of the PIN family of integral membrane auxin transporters (polarly localized in differentiated cells) led to insights into plant cell polarity. BASL, as a dually localized, nontransmembrane protein that exhibits activity and polarity during asymmetric cell divisions, represents an opportunity to explore polarity issues not accessible through study of any other known protein.
Genetic and physical interaction screens with BASL have been useful to expand this plant cell polarity module. Although such a module is in its infancy, we have isolated proteins whose functions we might expect; some, like BASL, provide the cell-type specificity and may act as scaffolds for plant-specific proteins that promote local growth, and the whole system will rely on general and conserved cellular processes. Continued screens and improved proteomics capabilities could identify additional components—and analysis of live-cell imaging could assign roles—for these new factors in cell polarity.
Stomatal Development and Function in Critical Plant Species in a Changing Climate
The collective activity of stomata drives global carbon and water cycles, but simultaneously, plant production of stomata is regulated by the global environment. How do plants sense the environment, and how do they alter their stomatal development in response? Although the sensors are still a mystery, many of the proteins we already study are parts of the response. In collaboration with Joseph Berry (Carnegie Institution for Science, Global Ecology), we are investigating the regulatory connection between environmental inputs and the activities of the stomatal transcription factors and signals. We are also using our ability to change stomatal numbers, arrangements, and functions to test whether these differences lead to changes in water use efficiency and drought tolerance under current and simulated climate change regimes. Understanding the mechanisms by which plants control the density and pattern of the structures that take in CO2 from the atmosphere may allow us to generate plants that can withstand altered climate or that can mitigate global warming by sequestering more atmospheric CO2.
Although Arabidopsis is a tractable system in which to identify genes and relationships required for stomatal development, neither crop improvements, nor energy security, nor climate change mitigation can be achieved without translation of these findings into other plants. Foremost in importance are the grasses; this family includes major food and fuel sources, and grasslands constitute major continental ecosystems. The study of stomatal development in the grasses is also biologically interesting because stomatal morphology, pattern, and ontogeny are significantly different than in broadleaf plants such as Arabidopsis. Our initial studies compared the functions of the homologs of Arabidopsis genes in the grasses. Now, thanks to genetic resources (mutagenized populations and natural accessions) created by our collaborator John Vogel (United States Department of AgricultureAgricultural Research Service), we are screening for potentially novel regulators of stomatal development in Brachypodium.
Grants from the National Institutes of Health, the National Science Foundation, and Bio-X (Stanford University) provided partial support for these projects.