Opposite polarity programs regulate asymmetric subsidiary cell divisions in grasses.

Grass stomata recruit lateral subsidiary cells (SCs), which are key to the unique stomatal morphology and the efficient plant-atmosphere gas exchange in grasses. Subsidiary mother cells (SMCs) strongly polarise before an asymmetric division forms a SC. Yet apart from a proximal polarity module that includes PANGLOSS1 (PAN1) and guides nuclear migration, little is known regarding the developmental processes that form SCs. Here, we used comparative transcriptomics of developing wild-type and SC-less bdmute leaves in the genetic model grass Brachypodium distachyon to identify novel factors involved in SC formation. This approach revealed BdPOLAR, which forms a novel, distal polarity domain in SMCs that is opposite to the proximal PAN1 domain. Both polarity domains are required for the formative SC division yet exhibit various roles in guiding pre-mitotic nuclear migration and SMC division plane orientation, respectively. Nonetheless, the domains are linked as the proximal domain controls polarisation of the distal domain. In summary, we identified two opposing polarity domains that coordinate the SC division, a process crucial for grass stomatal physiology.

Conservation and divergence of YODA MAPKKK function in regulation of grass epidermal patterning.

All multicellular organisms must properly pattern cell types to generate functional tissues and organs. The organized and predictable cell lineages of the Brachypodium leaf enabled us to characterize the role of the MAPK kinase kinase gene BdYODA1 in regulating asymmetric cell divisions. We find that YODA genes promote normal stomatal spacing patterns in both Arabidopsis and Brachypodium, despite species-specific differences in those patterns. Using lineage tracing and cell fate markers, we show that, unexpectedly, patterning defects in bdyoda1 mutants do not arise from faulty physical asymmetry in cell divisions but rather from improper enforcement of alternative cellular fates after division. These cross-species comparisons allow us to refine our understanding of MAPK activities during plant asymmetric cell divisions.

Mobile MUTE specifies subsidiary cells to build physiologically improved grass stomata.

Plants optimize carbon assimilation while limiting water loss by adjusting stomatal aperture. In grasses, a developmental innovation-the addition of subsidiary cells (SCs) flanking two dumbbell-shaped guard cells (GCs)-is linked to improved stomatal physiology. Here, we identify a transcription factor necessary and sufficient for SC formation in the wheat relative Brachypodium distachyon. Unexpectedly, the transcription factor is an ortholog of the stomatal regulator AtMUTE, which defines GC precursor fate in Arabidopsis The novel role of BdMUTE in specifying lateral SCs appears linked to its acquisition of cell-to-cell mobility in Brachypodium Physiological analyses on SC-less plants experimentally support classic hypotheses that SCs permit greater stomatal responsiveness and larger range of pore apertures. Manipulation of SC formation and function in crops, therefore, may be an effective approach to enhance plant performance.

Modulators of Stomatal Lineage Signal Transduction Alter Membrane Contact Sites and Reveal Specialization among ERECTA Kinases.

Signal transduction from a cell's surface to its interior requires dedicated signaling elements and a cellular environment conducive to signal propagation. Plant development, defense, and homeostasis rely on plasma membrane receptor-like kinases to perceive endogenous and environmental signals, but little is known about their immediate downstream targets and signaling modifiers. Using genetics, biochemistry, and live-cell imaging, we show that the VAP-RELATED SUPPRESSOR OF TMM (VST) family is required for ERECTA-mediated signaling in growth and cell-fate determination and reveal a role for ERECTA-LIKE2 in modulating signaling by its sister kinases. We show that VSTs are peripheral plasma membrane proteins that can form complexes with integral ER-membrane proteins, thereby potentially influencing the organization of the membrane milieu to promote efficient and differential signaling from the ERECTA-family members to their downstream intracellular targets.

Grasses use an alternatively wired bHLH transcription factor network to establish stomatal identity.

Stomata, epidermal valves facilitating plant-atmosphere gas exchange, represent a powerful model for understanding cell fate and pattern in plants. Core basic helix-loop-helix (bHLH) transcription factors regulating stomatal development were identified in Arabidopsis, but this dicot's developmental pattern and stomatal morphology represent only one of many possibilities in nature. Here, using unbiased forward genetic screens, followed by analysis of reporters and engineered mutants, we show that stomatal initiation in the grass Brachypodium distachyon uses orthologs of stomatal regulators known from Arabidopsis but that the function and behavior of individual genes, the relationships among genes, and the regulation of their protein products have diverged. Our results highlight ways in which a kernel of conserved genes may be alternatively wired to produce diversity in patterning and morphology and suggest that the stomatal transcription factor module is a prime target for breeding or genome modification to improve plant productivity.

Generation of signaling specificity in Arabidopsis by spatially restricted buffering of ligand-receptor interactions.

Core signaling pathways function in multiple programs during multicellular development. The mechanisms that compartmentalize pathway function or confer process specificity, however, remain largely unknown. In Arabidopsis thaliana, ERECTA (ER) family receptors have major roles in many growth and cell fate decisions. The ER family acts with receptor TOO MANY MOUTHS (TMM) and several ligands of the EPIDERMAL PATTERNING FACTOR LIKE (EPFL) family, which play distinct yet overlapping roles in patterning of epidermal stomata. Here, our examination of EPFL genes EPFL6/CHALLAH (CHAL), EPFL5/CHALLAH-LIKE1, and EPFL4/CHALLAH-LIKE2 (CLL2) reveals that this family may mediate additional ER-dependent processes. chal cll2 mutants display growth phenotypes characteristic of er mutants, and genetic interactions are consistent with CHAL family molecules acting as ER family ligands. We propose that different classes of EPFL genes regulate different aspects of ER family function and introduce a TMM-based discriminatory mechanism that permits simultaneous, yet compartmentalized and distinct, function of the ER family receptors in growth and epidermal patterning.

Regional specification of stomatal production by the putative ligand CHALLAH.

The problem of modulating cell fate programs to create distinct patterns and distributions of specialized cell types in different tissues is common to complex multicellular organisms. Here, we describe the previously uncharacterized CHALLAH (CHAL) gene, which acts as a tissue-specific regulator of epidermal pattern in Arabidopsis thaliana. Arabidopsis plants produce stomata, the cellular valves required for gas exchange, in virtually all aerial organs, but stomatal density and distribution differ among organs and along organ axes. Such regional regulation is particularly evident in plants mutant for the putative receptor TOO MANY MOUTHS (TMM), which produce excess stomata in leaves but no stomata in stems. Mutations in CHAL suppress tmm phenotypes in a tissue-specific manner, restoring stomatal production in stems while minimally affecting leaves. CHAL is similar in sequence to the putative stomatal ligands EPF1 and EPF2 and, like the EPFs, can reduce or eliminate stomatal production when overexpressed. However, CHAL and the EPFs have different relationships to TMM and the ERECTA (ER) family receptors. We propose a model in which CHAL and the EPFs both act through ER family receptors to repress stomatal production, but are subject to opposite regulation by TMM. The existence of two such ligand classes provides an explanation for TMM dual functionality and tissue-specific phenotypes.

Asymmetric cell divisions: a view from plant development.

All complex multicellular organisms must solve the problem of generating diverse and appropriately patterned cell types. Asymmetric division, in which a single mother cell gives rise to daughters with distinct identities, is instrumental in the generation of cellular diversity and higher-level patterns. In animal systems, there exists considerable evidence for conserved mechanisms of polarization and asymmetric division. Here, we consider asymmetric cell divisions in plants, highlighting the unique aspects of plant cell biology and organismal development that constrain the process, but also emphasizing conceptual and mechanistic similarities with animal asymmetric divisions.