ABSTRACT

SummaryLight-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 guardcell anion channels, through Ca 2+ -independent as well as Ca 2+ -dependent signalling pathways.

Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca2+ concentration. In Vicia faba, however, guard cell anion channels activate in a Ca2+-independent manner. Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca2+ concentration. ABA triggered transient rises in cytoplasmic Ca2+ in the majority of the guard cells (14 out of 19). In seven out of 14 guard cells, the change in cytoplasmic free Ca2+ closely matched the activation of anion channels, while the Ca2+ rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca2+ level. Even though ABA could activate anion channels in N. tabacum guard cells independent of a rise in the cytoplasmic Ca2+ concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca2+ in an ATP-dependent manner. Guard cells thus seem to have evolved both Ca2+-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca2+-independent pathway.

SummaryGuard cells respond to light through two independent signalling pathways. The first pathway is initiated by photosynthetically active radiation and has been associated with changes in the intercellular CO 2 concentration, leading to inhibition of plasma membrane anion channels. The second response is blue-light-specific and so far has been restricted to the activation of plasma membrane H + -ATPases. In a search for interactions of both signalling pathways, guard cells of Vicia faba and Arabidopsis thaliana were studied in intact plants. Vicia faba guard cells recorded in CO 2 -free air responded to blue light with a transient outward plasma membrane current that had an average peak value of 17 pA. In line with previous reports, changes in the current-voltage relation of the plasma membrane indicate that this outward current is based on the activation of H + -ATPases. However, when V. faba guard cells were blue-light-stimulated in air with 700 ll l )1 CO 2 , the outward current increased to 56 pA. The increase in current was linked to inhibition of S-type anion channels. Blue light also inhibited plasma membrane anion channels in A. thaliana guard cells, but not in the phot1 phot2 double mutant. These results show that blue light inhibits plasma membrane anion channels through a pathway involving phototropins, in addition to the stimulation of guard cell plasma membrane H + -ATPases.