A role for root carbonic anhydrase <scp>βCA4</scp> in the bicarbonate tolerance of <i>Arabidopsis thaliana</i>

Carbonic anhydrases (CAs) are the main enzymes handling bicarbonate in the different cell compartments. This study analyses the expression of CAs in roots of Arabidopsis thaliana demes differing in tolerance to bicarbonate: the tolerant A1_(C+) deme and the sensitive deme, T6_(C-). Exposure to 10 mM NaCl caused a transient depolarization of the root cell membranes, and in contrast, the supply of 10 mM NaHCO₃ caused hyperpolarization. This hyperpolarization was much stronger in A1_(C+) than in T6_(C-). Acetazolamide (AZ), a specific inhibitor of CAs, abolished the hyperpolarizing effect in A1_(C+), indicating the implication of CAs in this fast membrane response. The time-dependent (3 to 72 h) expression profiles of 14 CAs in roots of A1_(C+) and T6_(C-) exposed to either control (0 mM NaHCO₃, pH 5.9), or bicarbonate (10 mM NaHCO₃,pH 8.3) conditions revealed a bicarbonate specific upregulation of BCA4.1 (from 3 to 12 h) in A1_(C+). Contrastingly, in T6_(C-) BCA4.1 was downregulated by NaHCO₃. Exclusively in A1_(C+), the enhanced expression of BCA4.1 under bicarbonate was parallelled by an increase of PIP1,3, SLAH1, SLAH3, AHA2, and FRO2 gene expression levels. Under HCO₃ ^- exposure, a bca4 knockout mutant had a lower number of lateral roots, lower root diameters, and higher root lipid peroxidation than the WT. These results indicate that bicarbonate-induced root membrane hyperpolarization is the fast (minutes) initial signalling event in the tolerance response. This is followed by the specific upregulation of BCA4.1 and genes involved in H₂O and CO₂ transport, apoplast acidification, and iron acquisition.