Involvement of carbon monoxide produced by heme oxygenase in ABA-induced stomatal closure in Vicia faba and its proposed signal transduction pathway

Carbon monoxide (CO) has recently proven to be an important bioactive or signaling molecule in mammalian cells. Its effects are mainly mediated by nitric oxide (NO) and cyclic GMP (cGMP). In Vicia faba leaves, CO production and heme oxygenase (HO) activity, an important CO synthetic enzyme, are first reported to increase in response to ABA treatment, which could result in stomatal closure. Inter- estingly, ABA-induced stomatal closure in V. faba guard cells is partially blocked when the synthetic CO inhibitor ZnPP, or the CO/NO scavenger Hb is added. Furthermore, we show that, exogenously applied CO donor, hematin, and CO aqueous solution not only result in the enhancement of CO release, but also time-dependently induce stomatal closure, and the latter is mimicked by the application of an NO donor SNP. The above-mentioned stomatal closure effects are differentially reversed by the addition of tungstate, a potent inhibitor of NO synthetic enzyme nitrate reductase (NR), the specific NO scavenger cPTIO, ZnPP, or Hb. During treatment for 4 h, SNP, 0.01% CO aqueous solution or hematin significantly triggers NO synthesis, whereas cPTIO, or tungstate approximately fully inhibits NO fluorescence. Ad- ditionally, application of the GC inhibitor ODQ blocks CO-induced stomatal closure. This inhibition could be reversed when 8-Br-cGMP is added. Thus, the above results suggest that CO produced by HO is involved in ABA-induced stomatal closure, and NO and cGMP may function as downstream interme- diates in the CO signaling responsible for stomatal closure.