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Stomatal movement is regulated by changes in the volume of guard cells, thought to be mainly controlled by an osmo-regulatory system. In the present study, we examined the additional involvement of cytoskeletal events in the regulation of stomatal movement. Microtubules (MTs) in guard cells of Vicia faba L., grown under sunlight, were observed during the day and night by immunofluorescence microscopy. Cortical MTs began to be organized in a radial array at dawn and increased in numbers in the morning following the increase in the stomatal aperture size. Thereafter, MTs became localized near the nucleus and began to be destroyed from the evening to midnight, following the decrease in stomatal aperture size. These diurnal changes in MT organization were observed even two days after transfer from natural light condition to total darkness, and were accompanied by corresponding changes in stomatal aperture. The increase in stomatal aperture size in the early morning was inhibited by 50 microM propyzamide, which destroys cortical MTs in guard cells, whereas the decrease in aperture size in the evening was suppressed by 10 microM taxol, which stabilizes cortical MTs. These results suggest that radially-organized cortical MTs of guard cells may control diurnal stomatal movement.

To clarify the pathway and role of malate synthesis in guard cells, epidermal strips isolated from Vicia faba L. leaflets were treated with 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP), a specific inhibitor of phosphoenolpyruvate carboxylase (PEPC). When dark-closed stomata were illuminated, malate accumulated in guard cells and stomata opened; these were inhibited by 60% and 30%, respectively, by 5 mM DCDP treatment. When light-opened stomata were treated with DCDP, both malate level in guard cells and stomatal aperture decreased. Treatment with 5 mM DCDP partially inhibited CO2 incorporation into malate in guard cells. Treatment with mannitol at 0.4 M (osmotic stress) in the light increased malate level in guard cells and closed stomata. DCDP treatment decreased both malate level and stomatal aperture under stressed condition. These results show that malate synthesis in the light under both non-stressed and stressed conditions is dependent on PEPC activity. The extent of the decrease in malate level by DCDP treatment was larger under stressed condition than under nonstressed condition, suggesting that osmotic stress may enhance the activity of this pathway of malate synthesis which is induced by light. Role of malate synthesis in guard cells is discussed.

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