The continuing rise in atmospheric CO2 causes closing of stomatal pores in leaves and thus globally affects CO2 influx into plants, water use efficiency and leaf heat stress1–4. However, the CO2-binding proteins that control this response remain unknown. Moreover, the cell type that responds to CO2, mesophyll or guard cells, and whether photosynthesis mediates this response are matters of debate5–8. We demonstrate that Arabidopsis double mutant plants in the β-carbonic anhydrases, βCA1 and βCA4, display impaired CO2-regulation of stomatal movements and increased stomatal density, but retain functional abscisic-acid and blue-light responses.βCA-mediated CO2-triggered stomatal movements are not, in-first-order, linked to leaf-photosynthesis and can function in guard cells. Furthermore, guard cell βCA-over-expression plants exhibit enhanced water use efficiency. Guard cell-expression of mammalian αCAII complements ca1ca4 shows that carbonic anhydrase-mediated catalysis is an important mechanism for βCA-mediated CO2-induced stomatal closing and patch clamp analyses indicate that CO2/HCO3−transfers the signal to anion channel regulation. These findings, together with ht1-29 epistasis analysis demonstrate that carbonic anhydrases function early in the CO2 signalling pathway that controls gas-exchange between plants and the atmosphere.

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ORCID IDs: 0000-0003-0538-6646 (H.H.); 0000-0001-6675-273X (M.B.).Elevated carbon dioxide (CO 2 ) in leaves closes stomatal apertures. Research has shown key functions of the b-carbonic anhydrases (bCA1 and bCA4) in rapid CO 2 -induced stomatal movements by catalytic transmission of the CO 2 signal in guard cells. However, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) bCAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO 2 -regulated stomatal movements remains unknown. Here, we express fluorescently tagged CAs in guard cells of ca1ca4 double-mutant plants and show that the specific locations of bCA4 at the plasma membrane and bCA1 in native guard cell chloroplasts each can mediate rapid CO 2 control of stomatal movements. Localization and complementation analyses using a mammalian aCAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO 2 regulation of stomatal conductance. Mathematical modeling of cellular CO 2 catalysis suggests that the dynamics of the intracellular HCO 3 2 concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Moreover, modeling supports the notion that the intracellular HCO 3 2 concentration dynamics in guard cells are a key mechanism in mediating CO 2 -regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified.

To discover genes involved in nitric oxide (NO) metabolism, a genetic screen was employed to identify mutants defective in NO accumulation after treatment with the physiological inducer hydrogen peroxide. In wild-type Arabidopsis thaliana plants, NO levels increase eightfold in roots after H 2 O 2 treatment for 30 min. A mutant defective in H 2 O 2 -induced NO accumulation was identified, and the corresponding mutation was mapped to the prohibitin gene PHB3, converting the highly conserved Gly-37 to an Asp in the protein's SPFH domain. This point mutant and a T-DNA insertion mutant were examined for other NO-related phenotypes. Both mutants were defective in abscisic acid-induced NO accumulation and stomatal closure and in auxin-induced lateral root formation. Both mutants were less sensitive to salt stress, showing no increase in NO accumulation and less inhibition of primary root growth in response to NaCl treatment. In addition, light-induced NO accumulation was dramatically reduced in cotyledons. We found no evidence for impaired H 2 O 2 metabolism or signaling in the mutants as H 2 O 2 levels and H 2 O 2 -induced gene expression were unaffected by the mutations. These findings identify a component of the NO homeostasis system in plants and expand the function of prohibitin genes to include regulation of NO accumulation and NO-mediated responses.

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