Expanded roles and divergent regulation of FAMA in Brachypodium and Arabidopsis stomatal development.

Stomata, cellular valves found on the surfaces of aerial plant tissues, present a paradigm for studying cell fate and patterning in plants. A highly conserved core set of related basic helix-loop-helix (bHLH) transcription factors regulates stomatal development across diverse species. We characterized BdFAMA in the temperate grass Brachypodium distachyon and found this late-acting transcription factor was necessary and sufficient for specifying stomatal guard cell fate, and unexpectedly, could also induce the recruitment of subsidiary cells in the absence of its paralogue, BdMUTE. The overlap in function is paralleled by an overlap in expression pattern and by unique regulatory relationships between BdMUTE and BdFAMA. To better appreciate the relationships among the Brachypodium stomatal bHLHs, we used in vivo proteomics in developing leaves and found evidence for multiple shared interaction partners. We reexamined the roles of these genes in Arabidopsis thaliana by testing genetic sufficiency within and across species, and found that while BdFAMA and AtFAMA can rescue stomatal production in Arabidopsis fama and mute mutants, only AtFAMA can specify Brassica-specific myrosin idioblasts. Taken together, our findings refine the current models of stomatal bHLH function and regulatory feedback among paralogues within grasses as well as across the monocot/dicot divide.

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.

Single-cell resolution of lineage trajectories in the Arabidopsis stomatal lineage and developing leaf.

Dynamic cell identities underlie flexible developmental programs. The stomatal lineage in the Arabidopsis leaf epidermis features asynchronous and indeterminate divisions that can be modulated by environmental cues. The products of the lineage, stomatal guard cells and pavement cells, regulate plant-atmosphere exchanges, and the epidermis as a whole influences overall leaf growth. How flexibility is encoded in development of the stomatal lineage and how cell fates are coordinated in the leaf are open questions. Here, by leveraging single-cell transcriptomics and molecular genetics, we uncovered models of cell differentiation within Arabidopsis leaf tissue. Profiles across leaf tissues identified points of regulatory congruence. In the stomatal lineage, single-cell resolution resolved underlying cell heterogeneity within early stages and provided a fine-grained profile of guard cell differentiation. Through integration of genome-scale datasets and spatiotemporally precise functional manipulations, we also identified an extended role for the transcriptional regulator SPEECHLESS in reinforcing cell fate commitment.

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.