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

  • Nature volume 513, pages 246250 (11 September 2014)
  • doi:10.1038/nature13452
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Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development1,2,3; 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)6 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)7,8, 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 transcript 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.

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Proteomics Identifications Database

Data deposits

The raw data from three independent biological replicates in RNA-seq experiments have been deposited in the BioProject database under accession number PRJNA218542. The mass spectrometry proteomics data have been deposited in the Proteomics Identification Database (PRIDE) under accession numbers PXD000692, PXD000693 and PXD000956.


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We thank K. Knepper for conducting independent CO2-dependent stomatal development analyses. We thank A. Ries for help with generating the CA–YFP-fusion complementation lines. We thank D. Bergmann for providing the epfl6 mutant line and DNA constructs for MUTEpro::nucGFP expression; K. Torii for providing DNA constructs for MUTEpro::MUTE-GFP expression, erecta mutants and the oestradiol-inducible EPF constructs; T. Altmann for providing the sdd1-1 mutant; and M. Estelle, Y. Zhao, A. Stephan and M. Facette for comments on the manuscript. This project was funded by grants from the National Science Foundation (MCB0918220 and MCB1414339 to J.I.S. and IOS-1025837 to S.C.P.) and the National Institutes of Health (GM060396-ES010337 to J.I.S.), a BAYER-UC Discovery grant (J.I.S.) and a seed grant from the UCSD-SDCSB (GM085764) Systems Biology Center (C.B.E.). A grant from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy (DE-FG02-03ER15449) to J.I.S. funded complementation and localization analyses.

Author information

Author notes

    • Honghong Hu

    Present address: College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.


  1. Division of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA

    • Cawas B. Engineer
    • , Honghong Hu
    •  & Julian I. Schroeder
  2. Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, USA

    • Majid Ghassemian
  3. Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211, USA

    • Jeffrey C. Anderson
    •  & Scott C. Peck


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J.I.S. and C.B.E. designed experiments. C.B.E. conducted experiments (except for mass spectrometry analyses). C.B.E. generated GFP reporter expression and EPF overexpression plant lines. H.H. generated genetic constructs and transgenic plant lines for ca1 ca4 mutant YFP-fusion lines. Mass spectrometry analyses were conducted by M.G., J.C.A. and S.C.P. J.I.S. proposed the project. The manuscript was written by C.B.E. and J.I.S.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Julian I. Schroeder.

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