Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development(1–3); however, the key mechanisms mediating the perception and relay of the CO(2) signal to the stomatal development machinery remain elusive. To adapt CO(2) intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric CO(2) (ref. 4). To date, one mutant that exhibits deregulation of this CO(2)-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana β-carbonic anhydrase double mutants (ca1 ca4)(6) exhibit aninversion in their response to elevated CO(2), showing increased stomatal development at elevated CO(2) levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pathway involved in the regulation of CO(2)-controlled stomatal development by carbonic anhydrases. RNA-seq analyses of transcripts show that the extracellular pro-peptide-encoding gene EPIDERMAL PATTERNING FACTOR 2 (EPF2)(7,8), but not EPF1 (ref. 9), is induced in wild-type leaves but not inca1 ca4 mutant leaves at elevated CO(2) levels. Moreover, EPF2 is essential for CO(2) control of stomatal development. Using cell-wall proteomic analyses and CO(2)-dependent transcriptomic analyses, we identified a novel CO(2)-induced extracellular protease, CRSP (CO(2) RESPONSE SECRETED PROTEASE), as a mediator of CO(2)-controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric CO(2) elevation, including the carbonic-anhydrase-encoding genes CA1 and CA4 and the secreted protease CRSP, which cleaves the pro-peptide EPF2, in turn repressing stomatal development. Elucidation of these mechanisms advances the understanding of how plants perceive and relay the elevated CO(2) signal and provides a framework to guide future research into how environmental challenges can modulate gas exchange in plants

Molecular Framework of a Regulatory Circuit Initiating Two-Dimensional Spatial Patterning of Stomatal Lineage

Stomata, valves on the plant epidermis, are critical for plant growth and survival, and the presence of stomata impacts the global water and carbon cycle. Although transcription factors and cell-cell signaling components regulating stomatal development have been identified, it remains unclear as to how their regulatory interactions are translated into two-dimensional patterns of stomatal initial cells. Using molecular genetics, imaging, and mathematical simulation, we report a regulatory circuit that initiates the stomatal cell-lineage. The circuit includes a positive feedback loop constituting self-activation of SCREAMs that requires SPEECHLESS. This transcription factor module directly binds to the promoters and activates a secreted signal, EPIDERMAL PATTERNING FACTOR2, and the receptor modifier TOO MANY MOUTHS, while the receptor ERECTA lies outside of this module. This in turn inhibits SPCH, and hence SCRMs, thus constituting a negative feedback loop. Our mathematical model accurately predicts all known stomatal phenotypes with the inclusion of two additional components to the circuit: an EPF2-independent negative-feedback loop and a signal that lies outside of the SPCH•SCRM module. Our work reveals the intricate molecular framework governing self-organizing two-dimensional patterning in the plant epidermis