Carbonic anhydrases, EPF2 and a novel protease mediate C[O.sub.2] 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 C[O.sub.2] signal to the stomatal development machinery remain elusive. To adapt C[O.sub.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 C[O.sub.2] (ref. 4). To date, one mutant that exhibits deregulation of this C[O.sub.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 b-carbonic anhydrase double mutants (ca1 ca4) (6) exhibit an inversion in their response to elevated C[O.sub.2], showing increased stomatal development at elevated C[O.sub.2] levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pathway involved in the regulation of C[O.sub.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 in ca1 ca4 mutant leaves at elevated C[O.sub.2] levels. Moreover, EPF2 is essential for C[O.sub.2] control of stomatal development. Using cell-wall proteomic analyses and C[O.sub.2]-dependent transcriptomic analyses, we identified a novel C[O.sub.2]-induced extracellular protease, CRSP (C[O.sub.2] RESPONSE SECRETED PROTEASE), as a mediator of C[O.sub.2]-controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric C[O.sub.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 C[O.sub.2] signal and provides a framework to guide future research into how environmental challenges can modulate gas exchange in plants.