Release of Malate from Epidermal Strips during Stomatal Closure

Abstract

(4,6). The cell walls of the epidermis may also receive and store inorganic ions, although there is doubt whether their capacity suffices (5). The fate of the organic anions during stomatal closure has been studied after 14C labeling of guard cell contents (2). Epidermal samples were exposed to 4C02 for 2 min and then subjected to closing treatments. The analysis of the epidermes as well as of the liquid on which they floated indicated that guard cells may dispose of malate in three ways: (a) catabolism in the tricarboxylic acid cycle; (b) decarboxylation followed by gluconeogenesis and starch formation; and (c) release from the guard cells. However, it was estimated that only a small fraction of the total malate pool was labeled in these experiments. To determine the extent to which these three methods are employed by guard cells during stomatal closure, the fate of the whole malate pool must be examined. In particular, the importance of a specific release of malate was to be assessed because this mechanism would enable guard cells to reduce their turgor faster than metabolism of malic acid would allow (2). Changes in malate content were followed during stomatal closure in epidermal samples and in the water on which they floated. In most cases, stomatal closing was induced by addition of ABA to the water. described elsewhere (7). The second, third, and fourth fully expanded leaves were removed, rinsed with distilled H20, and cut into sections of I to 3 x 2 to 4 cm2. These sections were floated lower side up on distilled H20 in the light (85 w m-2 from mercury vapor lamps, General Electric H400 RDX 33-1) and C02-free air for 4 to 5 hr to allow stomata to open. Then the lower epidermis was removed in strips of about 5 x 10 to 15 mm2. Sixty to 80%o of the ordinary epidermal cells ruptured during peeling. The area of each strip was measured with a ruler. In four of five strips/experiment, stomatal apertures were measured under the microscope; then these strips were plunged into boiling 80%o ethanol to extract malate. The other strips were transferred to distilled H20 or solutions containing ABA. After incubation for various times, the strips were put on a microscope slide for the measurement of 20 to 25 stomatal apertures in each strip and then immediately extracted with boiling ethanol. The extracts were evaporated to dryness and analyzed for malate by enzymical oxidation, coupled to the reduction of NAD; the NADH formed was measured fluorimetrically (3; for details see 7). The solutions on which the strips had floated were also evaporated and assayed for malate.RESULTS AND DISCUSSION Epidermal strips of V. faba with open stomata were exposed in the light to a 0.1 mm solution of (±)-ABA for periods of 15 min to 2 hr (Fig. 1)

SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling

“…This is consistent with Cl − and malate efflux occurring in response to ABA 26,27 . We therefore applied whole-cell patch clamp techniques to characterize the functioning of S-type and R-type anion channel activities.…”

Malate. Jack of all trades or master of a few?

“…Similar to the situation described above for root exudation, malate has long been discussed as an important regulator of stomatal opening and is thought to be an integral part of the mechanism by which guard cells adjust their action in response to external CO 2 concentrations (Roefsema et al, 2002, Van Kirk andRaschke, 1978;Hedrich and Marten, 1993a, b;Hedrich et al, 1994;Raschke et al, 2003). However, in contrast to root exudation malate is the only carboxylic acid proposed to play a major role in this process.…”

Control of stomatal aperture

“…30,34,36,87,90 Recently a plasma membrane ABC malate uptake transporter was identified in guard cells. The gene encoding the ABC transporter family member ABCB14 (ABC transporter B family member 14), was identified and characterized in Arabidopsis plants.…”