(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)
Grapevine Red Blotch Virus is a major grapevine pathogens and is associated with reduced carbon assimilation and delayed berry ripening in Vitis vinifera L. Recent work suggests that the virus alters leaf carbon metabolism prior to emergence of visible symptoms. Therefore, diurnal and seasonal measurements were conducted to quantify changes in leaf carbon balance and to elucidate the chronology of symptom progression in leaves and fruit. Healthy and infected vines were assayed in a commercial vineyard during which leaf water relations, photosynthesis, and nonstructural carbohydrates were measured. Additionally, sugar and anthocyanin accumulation in the fruit were monitored at the end of the season to characterize the impact of the virus on ripening. Virus infection significantly reduced carbon assimilation pre- and postveraison, but the impact was more pronounced postveraison and during the afternoon when vine water status was the lowest. Similarly, virus infection significantly increased leaf starch concentration pre- and postveraison, but increased leaf starch in infected vines was detected two weeks prior to veraison. Virus infection had the greatest impact on obstructing leaf carbon export postveraison, especially during the afternoon. The virus had no impact on chlorophyll fluorescence, indicating there was no sustained photosystem impairment and suggesting that changes in chlorophyll fluorescence were a transient response to reduced carbon assimilation and export. This study provides evidence that reduced carbon export constitutes a feedback inhibition response to accumulation of leaf starch prior to the appearance of visible symptoms or impacts to ripening, which may aid earlier detection of the virus.
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