Silencing of NtMPK4 impairs CO2-induced stomatal closure, activation of anion channels and cytosolic Ca2+signals 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 CO2, 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 CO2 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 Ca2+ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca2+ than in those with steady Ca2+ levels. Both the darkness-induced anion channel activation and Ca2+ signals were repressed in NtMPK4-silenced guard cells. These data show that CO2 and darkness can activate anion channels in a Ca2+-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca2+ concentration. NtMPK4 plays an essential role in CO2- and darkness-induced activation of guard-cell anion channels, through Ca2+-independent as well as Ca2+-dependent signalling pathways.