Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells.

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of NtMPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of NtMPK4-silenced plants do not close in elevated atmospheric CO(2), and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO(2) activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in NtMPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca(2+) concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca(2+) than in those with steady Ca(2+) levels. Both the darkness-induced anion channel activation and Ca(2+) signals were repressed in NtMPK4-silenced guard cells. These data show that CO(2) and darkness can activate anion channels in a Ca(2+)-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca(2+) concentration. NtMPK4 plays an essential role in CO(2)- and darkness-induced activation of guard-cell anion channels, through Ca(2+)-independent as well as Ca(2+)-dependent signalling pathways.