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Stomata of Commelina leaves pre-opened by incubation in moist air were found to close within 30 min when supplied with abscisic acid (ABA) via the transpiration stream. Radioactive ABA had similar effects, but allowed the distribution of the compound within the leaf to be measured and correlated with stomatal movements to give estimates of the sensitivity of Commelina stomata. On a whole-leaf basis, less than 163 fmol ABA per mm(2) leaf area were present at the time of complete stomatal closure. This was close to other published estimates. By taking epidermal (14)C measurements, however, it was possible to increase the accuracy of the estimate on the assumption that only ABA present in the epidermis was physiologically active. Thus, less than 235 amol ABA for stomatal complex were present at complete closure, and statistically significant narrowing of the stomatal aperture had occurred when between 12.6 and 45.4 amol per complex were present. The distribution of ABA within the epidermal tissue after transpiration-stream application was studied using microautoradiography, and the compound appeared to have accumulated within the stomatal complex.

Guard-cell protoplasts were isolated by enzymic digestion of the epidermis peeled from the abaxial surface of leaves from Commelina communis L. The protoplasts were separated from mesophyll-cell protoplasts and other contaminants by density-gradient centrifugation, and the purity of the preparations carefully and quantitatively assessed by light microscopy. The preparations of guard-cell protoplasts were then compared with mesophyll-cell protoplasts in terms of the activity of photosystem II as assessed by a) the light-induced evolution of oxygen under both steady-state and flashing light and b) the characteristics of photosystem-II chlorophyll fluorescence. In all experiments, clear photosystem-II activity was found in guard-cell protoplasts, although some subtle distinctions between guard-cell and mesophyll-cell protoplasts were found. The contribution of any contaimination by mesophyll-cell chlorophyll to guard-cell-protoplast signals was estimated to be less than 3% in all cases. The results indicate that photosystem II is present and active in guard cells of Commelina.

Closure of stomata by abscisic acid (ABA) was studied by floating leaf epidermal strips of Commelina communis L. in PIPES buffer (pH 6.8) containing a range of KCl concentrations. Control apertures were greatest at high concentrations of the salt, and the effects of ABA, in terms of closure, were most pronounced below 100 mol m(-3) KCl. Stomata opened on strips floated on buffer plus 50 mol m(-3) KCl and closed within 10 min when transferred to the same medium plus 0.1 mol m(-3) ABA. [2-(14)C]ABA was used to study uptake and distribution of the hormone by the epidermal strips. It was calculated that no more than 6 fmol ABA were present per stomatal complex at the time of closure, although uptake continued thereafter. Microautoradiography indicated that radioactivity from [2-(14)C]ABA accumulated in the stomatal complex at or near the guard cells within 20 min. TLC was used to examine the state of the label after 1 h incubation. Efflux of label from preincubated tissue appeared to occur in three phases (t1/2=7.2 s, 4.0 min, 35.2 min). Efflux was correlated with stomatal re-opening. The results confirm that ABA can accumulate in the epidermis of C. communis.