title: Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development.
abstract: Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; however, the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 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 CO2 (ref. 4). To date, one mutant that exhibits deregulation of this CO2-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) exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pathway involved in the regulation of CO2-controlled stomatal development by carbonic anhydrases. RNA-seq analyses of transcripts show that the extracellular pro-peptide-encoding gene EPIDERMAL PATTERNING FACTOR 2 (EPF2), but not EPF1 (ref. 9), is induced in wild-type leaves but not in ca1 ca4 mutant leaves at elevated CO2 levels. Moreover, EPF2 is essential for CO2 control of stomatal development. Using cell-wall proteomic analyses and CO2-dependent transcriptomic analyses, we identified a novel CO2-induced extracellular protease, CRSP (CO2 RESPONSE SECRETED PROTEASE), as a mediator of CO2-controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric CO2 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 CO2 signal and provides a framework to guide future research into how environmental challenges can modulate gas exchange in plants.
authors.affiliation: Division of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Cawas B
authors.initials: CB
authors.lastname: Engineer
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Majid
authors.initials: M
authors.lastname: Ghassemian
authors.affiliation: Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Anderson
authors.affiliation: Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Peck
authors.affiliation: 1] Division of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA [2] College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Honghong
authors.initials: H
authors.lastname: Hu
authors.affiliation: Division of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Julian I
authors.initials: JI
authors.lastname: Schroeder
mesh_terms: D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism* / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D009154:Mutation; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D054046:Plant Stomata / Q000254:growth & development*; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; C542689:EPF2 protein, Arabidopsis; D014157:Transcription Factors; D002245:Carbon Dioxide; C000591840:CRSP protein, Arabidopsis; D010447:Peptide Hydrolases; D002256:Carbonic Anhydrases