Form(ation) and function of plant stomata

Plant stomata are tiny “breathing” pores on the leaf surface that take up carbon dioxide (CO₂), which with the help of sunlight is turned into the sugars we eat and the oxygen we breathe. At the same time, the stomata release water vapor, which needs to be tightly regulated and balanced with CO₂ uptake to conserve as much water as possible. The water that transpires through stomata make up 50% of all the water that evaporates from our planet. Stomata cycle twice the atmospheric water content per year and fix 20% of the atmospheric carbon dioxide (CO₂). Stomatal development is controlled by a suite of bHLH transcription factors and a sophisticated regulatory module that properly patterns stomata and adjusts the number of stomata per leaf. Therefore, plant stomata offer an outstanding developmental and physiological model system that links plant fitness with global climate dynamics.

To make gas exchange more water-use-efficient  some plant families form innovative stomatal morphologies. Grasses like the model system Brachypodium distachyon and cereal crops like rice, maize and wheat, for example, make four-celled stomatal complexes by adding two intimately connected, lateral subsidiary cells (or “helper cells”) to the central guard cells. The subsidiary cells are a developmental innovation that allows grasses to “breathe” more efficiently and therefore save water.  The Raissig Lab uses grass models and other, novel plant model systems with diverse stomatal morphologies to study (1) how different subsidiary cells are formed, (2) if and how they are of functional relevance, and (3) how such different stomatal morphologies could arise in an evolutionary context.

 

Development and function of grass subsidiary cells

We previously discovered that the mobile transcription factor BdMUTE specifies subsidiary cells in the model grass and wheat relative Brachypodium distachyon (Raissig et al. 2017 Science). The mutant bdmute fails to recruit subsidiary cells and forms two-celled, eudicot-like stomata instead (see image) that are slower to open and close and fail to open as wide as four-celled wild-type stomata. We sequenced the transcriptome of both wild-type and bdmute mutant leaf zones and discovered genes absent or strongly downregulated in the mutant that are potentially associated with subsidiary cell formation and function. Reverse genetic approaches (e.g. mutant collection and CRISPR-based gene editing), analysis of reporter genes, overexpression constructs and leaf-level gas exchange measurements will determine the differentially expressed candidates’ roles in subsidiary cell formation and function. Finally, we are using guard-cell-specific and subsidiary-cell-specific reporter lines to isolate and sequence pools of cell-type-specific libraries and determine the molecular signatures of these two cell lineages at different developmental stages.

 

Epidermal development of grass leaves in time and space

The grass epidermis offers a highly accessible and tractable developmental gradient with the young, undifferentiated cells at the bottom and the mature, differentiated cells at the top of the leaf. We want to perform high-resolution time-lapse imaging of early epidermal development in Brachypodium to understand the dynamics of SC recruitment as well as general rules and patterns of early grass epidermal development. Dual color imaging of a ubiquitously expressed plasmamembrane marker  together with tagged stomatal transcription factors, cell cycle regulators or cytoskeleton markers will allow us to describe and model aspects of grass epidermal development, such as, where and how cells divide, elongate and differentiate. This compendium of rules and patterns will then be used to screen for more subtle stomatal morphology and patterning mutants in another forward genetic screen. In addition, general epidermal patterning or fate mutants might be discovered in the screen.

 

Stomatal development and physiology in C₄ and CAM plants

Our lab is establishing novel stomatal model systems with distinct stomatal morphologies and physiological lifestyles. The millet Setaria viridis produces more triangular subsidiary cells and is a C₄ plant that uses anatomical leaf features (bundle sheath cells) to concentrate carbon dioxide and optimize photosynthesis. We hope to describe whether MUTE’s role is conserved in grasses, how the different subsidiary cell shape arrises, and, finally, how important subsidiary cells are in a C₄ grass.

To exploit the morphological spectrum of subsidiary cells and to cover the third photosynthetic lifestyle Crassulacean acid metabolism (CAM), we are establishing a Kalanchoë sp. as developmental model system. The Kalanchoë genus produces stomata with subsidiary-like cells arranged in a circle and likely of a different origin than those in grasses. CAM plants like Kalanchoë open stomata for CO₂ uptake at night only and photosynthesize during the day with closed stomata, which makes CAM plants extremely water efficient. Functionally, subsidiary-like cells in Kalanchoë are an enigma, since fast responsiveness seems not to be required for a diurnal stomatal lifestyle. We will perform candidate gene editing using CRISPR/Cas9 systems, cell-type specific transcriptomics and reporter gene analysis to understand how Kalanchoë forms subsidiary-like cells and if and how they are functionally relevant.