ABA contents in the guard-cell symplast and guard-cell apoplast are not correlated with stomatal aperture size under three conditions of water sufficiency

Guard cells respond to environmental stimuli by opening and closing stomata, which balance CO2 uptake and water conservation. Stomatal closure under water deficiency and the involvement of abscisic acid (ABA) in this response are well-known. However, whether ABA plays a role in stomatal regulation under some water-sufficient conditions, such as diurnal changes, humidity shift (i.e. transpiration-rate change) and brief flooding, is not clear. Using an enzyme-linked immunosorbent assay (ELISA) with sub-femtomole sensitivity for ABA assays, we studied the relationships of stomatal aperture size with the ABA contents in the symplast and apoplast of guard cells, as well as those in the leaf and the leaf apoplast in Vicia faba under the following three conditions. (1) Diurnal changes. Stomata opened in the morning, reached a maximum opening at 1400 h, and closed at 1800 h. Neither the leaf nor the leaf-apoplastic ABA content strongly correlated with stomatal aperture sizes. The ABA contents of the guard-cell compartments did not change over the course of the day, providing evidence that ABA is not involved in diurnal stomatal regulation. (2) Humidity-induced transpiration-rate changes. The transpiration rate of intact plants and that of detached leaves infused with 1muM ABA was decreased by shifting RH from 60% to 90%. However, the ABA contents of the four compartments were not changed by this humidity shift, in spite of an increase of 2--3 mum in stomatal aperture sizes. Thus, the guard-cell-apoplastic ABA content is not affected by transpiration rate, and ABA may not participate in the stomatal response to transpiration rate. (3) Brief flooding. Stomata closed after brief (4-h) flooding, when the leaf and the leaf apoplastic ABA increased 2--3 fold and the xylem pH increased 0.2 pH units. The leaf ABA increase did not correlate strongly with stomatal aperture size and the xylem ABA delivery rate remained unchanged. The ABA contents in the guard-cell compartments of flooded plants were not different from those of non-flooded plants. Therefore, ABA may not be an initiator of stomatal closure under brief flooding, and xylem alkalinization probably does not induce leaf ABA redistribution to guard cells.