Differentiation of specialized cell types in multicellular organisms requires orchestrated actions of cell fate determinants. Stomata, valves on the plant epidermis, are formed through a series of differentiation events mediated by three closely related basic-helix-loop-helix proteins: SPEECHLESS (SPCH), MUTE, and FAMA. However, it is not known what mechanism coordinates their actions. Here, we identify two paralogous proteins, SCREAM (SCRM) and SCRM2, which directly interact with and specify the sequential actions of SPCH, MUTE, and FAMA. The gain-of-function mutation in SCRM exhibited constitutive stomatal differentiation in the epidermis. Conversely, successive loss of SCRM and SCRM2 recapitulated the phenotypes of fama, mute, and spch, indicating that SCRM and SCRM2 together determined successive initiation, proliferation, and terminal differentiation of stomatal cell lineages. Our findings identify the core regulatory units of stomatal differentiation and suggest a model strikingly similar to cell-type differentiation in animals. Surprisingly, map-based cloning revealed that SCRM is INDUCER OF CBF EXPRESSION1, a master regulator of freezing tolerance, thus implicating a potential link between the transcriptional regulation of environmental adaptation and development in plants.

Valves on the plant epidermis called stomata develop according to positional cues, which likely involve putative ligands (EPIDERMAL PATTERNING FACTORS [EPFs]) and putative receptors (ERECTA family receptor kinases and TOO MANY MOUTHS [TMM]) in Arabidopsis. Here we report the direct, robust, and saturable binding of bioactive EPF peptides to the ERECTA family. In contrast, TMM exhibits negligible binding to EPF1 but binding to EPF2. The ERECTA family forms receptor homomers in vivo. On the other hand, TMM associates with the ERECTA family but not with itself. While ERECTA family receptor kinases exhibit complex redundancy, blocking ERECTA and ERECTA-LIKE1 (ERL1) signaling confers specific insensitivity to EPF2 and EPF1, respectively. Our results place the ERECTA family as the primary receptors for EPFs with TMM as a signal modulator and establish EPF2–ERECTA and EPF1–ERL1 as ligand–receptor pairs specifying two steps of stomatal development: initiation and spacing divisions.

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca²⁺ binding EF-hand motifs, possessed Ca²⁺-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca²⁺-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca²⁺-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca²⁺. Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca²⁺-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

Patterning of stomata, valves on the plant epidermis, requires the orchestrated actions of signaling components and cell-fate determinants. To understand the regulation of stomatal patterning, we performed a genetic screen using a background that partially lacks stomatal signaling receptors. Here, we report the isolation and characterization of chorus (chor), which confers excessive proliferation of stomatal-lineage cells mediated by SPEECHLESS (SPCH). chor breaks redundancy among three ERECTA family genes and strongly enhances stomatal patterning defects caused by loss-of-function in TOO MANY MOUTHS. chor seedlings also exhibit incomplete cytokinesis and growth defects, including disruptions in root tissue patterning and root hair cell morphogenesis. CHOR encodes a putative callose synthase, GLUCAN SYNTHASE-LIKE 8 (GSL8), that is required for callose deposition at the cell plate, cell wall and plasmodesmata. Consistently, symplastic macromolecular diffusion between epidermal cells is significantly increased in chor, and proteins that do not normally move cell-to-cell, including a fluorescent protein-tagged SPCH, diffuse to neighboring cells. Such a phenotype is not a general trait caused by cytokinesis defects. Our findings suggest that the restriction of symplastic movement might be an essential step for the proper segregation of cell-fate determinants during stomatal development.