The MAPK substrate MASS proteins regulate stomatal development in Arabidopsis

Author summary Stomata surrounded by guard cells are breathing pores in the plant epidermis, where they open to allow gas exchange and close to restrict water loss. The production and patterning of stomata in the model plant Arabidopsis provide an ideal genetic and cell biological system for studying the molecular mechanisms underlying developmental program and plasticity in responding to environmental changes. The MAPK cascades are ubiquitous signaling modules in eukaryotes. They regulate diverse cellular programs by relaying extracellular signals to intracellular regulators. In the model plant Arabidopsis, MAPK 3 and 6 were found to phosphorylate several protein substrates in the nucleus and cytoplasm to regulate stomatal development and patterning. In this study, we report that a group of new MAPK substrates, the MASS proteins, function at the plasma membrane to regulate stomatal production and patterning in Arabidopsis. Thus, the output of MAPK signaling in the regulation of stomatal development is diverged by differentially localized substrates, suggesting that the concerted activities of MAPK substrates fine-tune stomatal development to ultimately improve plant adaptability to the changing environment. Stomata are specialized pores in the epidermis of the aerial parts of a plant, where stomatal guard cells close and open to regulate gas exchange with the atmosphere and restrict excessive water vapor from the plant. The production and patterning of the stomatal lineage cells in higher plants are influenced by the activities of the widely-used mitogen-activated protein kinase (MAPK) signaling components. The phenotype caused by the loss-of-function mutations suggested pivotal roles of the canonical MAPK pathway in the suppression of stomatal formation and regulation of stomatal patterning in Arabidopsis, whilst the cell type-specific manipulation of individual MAPK components revealed the existence of a positive impact on stomatal production. Among a large number of putative MAPK substrates in plants, the nuclear transcription factors SPEECHLESS (SPCH) and SCREAM (SCRM) are targets of MAPK 3 and 6 (MPK3/6) in the inhibition of stomatal formation. The polarity protein BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) is phosphorylated by MPK3/6 for localization and function in driving divisional asymmetries. Here, by functionally characterizing three MAPK SUBSTRATES IN THE STOMATAL LINEAGE (MASS) proteins, we establish that they are plasma membrane-associated, positive regulators of stomatal production. MPK6 can phosphorylate the MASS proteins in vitro and mutating the putative substrate sites interferes the subcellular partition and function of MASS in planta. Our fine-scale domain analyses identify critical subdomains of MASS2 required for specific subcellular localization and biological function, respectively. Furthermore, our data indicate that the MASS proteins may directly interact with the MAPKK Kinase YODA (YDA) at the plasma membrane. Thus, the deeply conserved MASS proteins are tightly connected with MAPK signaling in Arabidopsis to fine-tune stomatal production and patterning, providing a functional divergence of the YDA-MPK3/6 cascade in the regulation of plant developmental processes.