Reconstitution of CO₂ Regulation of SLAC1 Anion Channel and Function of CO₂-Permeable PIP2;1 Aquaporin as CARBONIC ANHYDRASE4 Interactor

Abstract

Dark respiration causes an increase in leaf CO 2 concentration (Ci), and the continuing increases in atmospheric [CO 2 ] further increases Ci. Elevated leaf CO 2 concentration causes stomatal pores to close. Here, we demonstrate that high intracellular CO 2 /HCO 3 2 enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the bCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO 2 permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO 2 -and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of bCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO 2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO 2 permeability and extracellular CO 2 regulation of SLAC1 activity. These findings identify the CO 2 -permeable PIP2;1 as key interactor of bCA4 and demonstrate functional reconstitution of extracellular CO 2 signaling to ion channel regulation upon coexpression of PIP2;1, bCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO 2 regulation of S-type anion channels.

Natural Variation in Arabidopsis Cvi-0 Accession Reveals an Important Role of MPK12 in Guard Cell CO2 Signaling

“…(Left guard cell) CO 2 enters guard cells through the PIP2;1 aquaporin [20] and is converted to bicarbonate by carbonic anhydrases βCA4 and βCA1. The mechanism by which bicarbonate is sensed in guard cells still needs to be resolved; nevertheless, it is likely that in elevated CO 2 conditions, activation of MPK12 and MPK4 leads to inhibition of HT1, and this enables activation of slow-type anion channel SLAC1 by OST1 [15].…”

“…laevis oocytes as a heterologous expression system to reconstitute bicarbonate-induced activation of the SLAC1 anion channel [19,20]. Tian et al [19] reported that a multidrug and toxic compound extrusion (MATE)-type transporter RHC1 functions as a bicarbonate-sensing component that inactivates HT1 and promotes SLAC1 activation by OST1.…”

“…Isolation of a dominant HT1 allele, ht1-8D , revealed that HT1 may directly inhibit OST1- and GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1 (GHR1)-induced activation of SLAC1 [18]. Bicarbonate-induced activation of SLAC1 has been reconstituted in Xenopus laevis oocytes [19,20]. The pathway was shown to consist of RESISTANT TO HIGH CARBON DIOXIDE 1 (RHC1), HT1, OST1, and SLAC1 [19], while more recently the importance of CARBONIC ANHYDRASE 4 (βCA4), aquaporin PIP2;1, OST1, and SLAC1 was demonstrated [20].…”

“…Bicarbonate-induced activation of SLAC1 has been reconstituted in Xenopus laevis oocytes [19,20]. The pathway was shown to consist of RESISTANT TO HIGH CARBON DIOXIDE 1 (RHC1), HT1, OST1, and SLAC1 [19], while more recently the importance of CARBONIC ANHYDRASE 4 (βCA4), aquaporin PIP2;1, OST1, and SLAC1 was demonstrated [20]. Although guard cells are perhaps the best characterized single cell signaling system in the plant kingdom, there are still large gaps in our understanding of how CO 2 signaling in guard cells is regulated and by which mechanism CO 2 might regulate plant water management and WUE [5,21,22].…”

The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis

“…In the CO 2 -specific signaling pathway, a recent study has shown that the CO 2 -permeable PIP2;1 aquaporin protein is a key interactor of the b-carbonic anhydrase 4 that catalyzes a reaction converting CO 2 into bicarbonate and protons, and mediates downstream CO 2 signaling (Wang et al, 2016). The participation of protein kinases in bicarbonate activation of S-type anion channels has been shown in guard cells (Hashimoto et al, 2006;Xue et al, 2011;Tian et al, 2015), and a new study also shows that SLAC1 is a bicarbonate sensor in CO 2 regulation of SLAC1 activation (Wang et al, 2016). Taken together, these results suggest that multiple pathways could be involved in the mediation of CO 2 responses in vivo, consistent with a recent study on carbonic anhydrase functions (Hu et al, 2015).…”