Original objectives (revisions from original proposal are highlighted) 1. Elucidate the direct effects O3 and H2O2 on guard cell function, utilizing assays of stomatal response in isolated epidermal peels and whole cell gas exchange. 2. Determine the mechanistic basis of O3 and H2O2 effects on the plasma membrane through application of the electrophysiological technique of patch clamping to isolated guard cells. 3. Determine the relative sensitivity of Israeli cultivars of economically important crops to O3 and determine whether differential leaf conductance responses to O3 can explain relative sensitivity to the air pollutant: transfer of technological expertise to Israel. Background to the topic For a long time O3 has been known to reduce gas exchange in plants; it has however been unclear if O3 can affect the stomatal complex directly. Ion channels are essential in stomatal regulation, but O3 has never before been shown to affect these directly. Major conclusions, solution, achievements 1. Ozone inhibits light-induced stomatal opening in epidermal peels isolated from Vicia faba, Arabidopsis thaliana and Nicotiana tabacum in V. faba plants this leads to reduced assimilation without a direct effect on the photosynthetic apparatus. Stomatal opening is more sensitive to O3 than stomatal closure. 2. Ozone causes inhibition of inward K+ channels (involved in stomatal opening) while no detectable effect is observed o the outward K+ channels (stomatal closure). 3. Hydrogen peroxide inhibits stomatal opening and induces stomatal closure in epidermal peels isolated from Vicia faba. 4. Hydrogen peroxide enhances stomatal closure by increasing K+ efflux from guard cells via outward rectifying K+ channels. 5. Based on epidermal peel experiments we have indirectly shown that Ca2+ may play a role in the guard cell response to O3. However, direct measurement of the guard cell [Ca2+]cyt did not show a response to O3. 6. Three Israeli cultivars of zucchini, Clarita, Yarden and Bareqet, were shown to be relatively sensitive to O3 (0.12 ml1-1 ). 7. Two environmentally important Israeli pine species are adversely affected by O3, even at 0.050 ml1-1 , a level frequently exceeded under local tropospheric conditions. P. brutia may be better equipped than P. halepensis to tolerate O3 stress. 8. Ozone directly affects pigment biosynthesis in pine seedlings, as well as the metabolism of O5 precursors, thus affecting the allocation of resources among various metabolic pathways. 9. Ozone induces activity of antioxidant enzymes, and of ascorbate content i the mesophyll and epidermis cells of Commelina communis L. Implications, both scientific and agricultural We have improved the understanding of how O3 and H2O2 do affect guard cell and stomatal function. We have shown that economical important Israeli species like zucchini and pine are relatively sensitive to O3.
K+ transport properties of K+ channels in the plasma membrane of Vicia faba guard cells.
