3Abbreviation: NCE, net carbon exchange. important for carbon balance, is closely controlled. Regulation of the metabolism of starch in source leaves is not ...
Plant Physiol. (1984) 76, 763-768 0032-0889/84/76/0763/06/$01.00/0
Effects of Decreased Net Carbon Exchange on Carbohydrate Metabolism in Sugar Beet Source Leaves1 Received for publication February 13, 1984 and in revised form July 26, 1984
THEODORE C. FOX2 AND DONALD R. GEIGER* Department of Biology, University of Dayton, Dayton, Ohio 45469 ABSTRACT The relationship between CO2 concentration and starch synthesis and degradation was studied by measuring leaf starch content and disappearance of '4C-starch. At a concentration of 340 microliters CO2 per liter, starch accumulated without degradation of previously synthesized starch. Degradation of starch began when CO2 concentration was lowered, but its synthesis continued. At 120 microliters CO2 per liter rates of synthesis and degradation were equal. Even at the CO2 compensation point, synthesis of starch continued. Concomitant starch synthesis and mobilization supported export from the leaf. Changes in starch metabolism that occur when photosynthesis is COrlimited provide a means to study regulation of starch metabolism and carbon allocation in translocating leaves.
important for carbon balance, is closely controlled. Regulation of the metabolism of starch in source leaves is not understood well. Because starch is the major source of carbon during the night (7) its accumulation likely will decrease only with a relatively serious disruption of carbon supply. Whereas adjustment of starch accumulation to lowered NCE may be made directly by lowering synthesis rate, concurrent synthesis and breakdown of starch have been reported in illuminated chloroplasts (23) and intact plants (6, 16). In this study we have investigated the relation between NCE and starch accumulation and degradation in exporting leaves of sugar beet, a species that utilizes starch as its main carbohydrate reserve.
MATERIALS AND METHODS Plant Material. Sugar beet plants (Beta vulgaris L., Klein E type, multigerm) were cultured in a mixture of sand and peat moss-vermiculite (1:1) in a controlled environmental chamber Translocation of assimilates is controlled by regulating export (14 h d at 24°C, 17°C). Photon flux density was 340 ,mol m-2 from source leaves and by partitioning of the translocated com- s-I PAR at leaf blade level. We found that NCE at this irradiance pounds among sinks (9). Maintenance of carbon balance among was approximately 65% of the rate for sugar beet plants raised plant organs is attained by control of carbon flow within and out and measured at 1000 ,mol m-2 s-' (J. Bunce, Beltsville, personal of photosynthesizing leaves. Carbon metabolism regulates the communication). Plants were watered with a nutrient solution availability of carbon compounds to various processes, including (22). export, thereby allowing the plant to adjust its physiological state Labeling Procedure. Two adjacent source leaves, each in an in a changing environment. individual chamber, were exposed to a circulating atmosphere. Products of photosynthesis destined for export are partitioned CO2 was metered into the closed system to hold concentration among several source leaf pools (4, 5, 7, 9). In sugar beet, the to within 5% of the desired level, with or without '4C02 of major mesophyll carbohydrate pools are sucrose, hexoses, and constant specific radioactivity (10, 11). Labeling was carried out starch (7). Regulation of allocation among these pools provides for an entire day and for 4 to 6 h on the 2nd day to allow starch sufficient carbon reserves to support translocation during periods to become extensively labeled. Usually a chase period with of low or zero NCE3 while maintaining a pool of sucrose for unlabeled CO2 followed the second labeling period. Irradiance immediate export of carbon from the leaf. level was the same as that under which the plants were grown. A Many plants, including barley (12), use sucrose as a major rotary displacement transducer (Schaevitz R30D) in the labeling form of storage carbohydrate. Gordon et al. (12) have character- chamber monitored leaf thickness as a measure of water status. ized the pattern of carbohydrate allocation in barley. During the Sampling Procedure. Sampling began at the end of the second day, leaf sucrose increases 2- to 3-fold (dry weight basis) and is labeling period and continued for 4 to 8 h. At each sampling also exported. Starch accumulates to a smaller extent. At night, time two or four punches per blade were removed, with a leaf sucrose export occurs for 8 to 9 h before starch mobilization combined area of 0.34 or 0.69 cm2, respectively. Care was taken begins (13, 14), possibly in response to partial depletion of the so that areas sampled were not isolated from a major vein by sucrose pool. previous samples. By contrast, starch is the major carbohydrate reserve in leaves The carbohydrate content ofdifferent leaves on the same plant of sugar beet, with sucrose being relatively less important in this is too different to be compared and allowance must be made for regard. At night, starch mobilization maintains export of sucrose this fact. The content in leaves from different plants is even more from source leaves in these plants. The size of reserve carbohydrate pools, which appears to be 1 Supported by Grants PCM-8008720 and PCM-8303957 from the
variable. Carbohydrate levels in sets of two disks sampled from the same leaf allow changes to be followed over a period of 30 to 60 min. To evaluate variability, standard deviations for starch content were calculated for three groups of data, each consisting of National Science Foundation. 2 Present address: Department of Horticulture and Landscape Archi- 15 pairs of punches from a different leaf. Values of 2.6, 9.1, and tecture, Washington State University, Pullman, Washington 991646414. 4.8% of the mean were found for samples taken over a period short enough that the starch content could not increase markedly. 3Abbreviation: NCE, net carbon exchange. 763
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'Lc"fw""X AND GEIGER
As a consequence of the variability among plants, representative curves are shown for some figures. Values for standard deviations or ranges obtained by averaging data from several plants will include a large among-plants component. Overall standard deviations of data for carbohydrate levels averaged for leaves from three plants were on the order of 40% while those of the three component curves were on the order of 5% of the mean. Extraction. Immediately upon removal from the leaf blade, punches were extracted by refluxing in chloroform-methanol (4:1) at 65°C until the disks were white. The disks were rinsed and refluxed for an additional 30 min with 200 Ml 80% ethanol at 65C. The organic phase from the combined extracts was discarded. The water phase was dried at 30°C and then was resuspended in 400 ;d deionized H20 for the carbohydrate determinations. Contact images of the extracted, wet disks were made on x-ray film and used to measure area. The disks were dried at 80C for 2 to 3 d and weighed with a Cahn RG microbalance. Starch was determined by the method of Outlaw and Manchester (19) after maceration of the tissue (8). Disks were incubated for 10 to 12 h in 400 Ml 0.2 N KOH containing 100 mM ethanol at room temperature.The brei resulting from a 2-min homogenization was heated to 80°C for 2 h. Volume was brought to 500 Ml with water and the pH was adjusted with glacial acetic acid. Starch was assayed as glucose that was released by adding 0.25 mg amyloglucosidase to each sample and incubating the mixture for 10 h. Glucose, fructose, and sucrose were determined with an enzymic assay based on the direct spectrofluorometric measure of NADPH (17, method B). For glucose determination, 20 MAl of sample was added to 2 ml of a reaction mixture containing 50 mM imidoazole (pH 6.9 with HCI), 1 mM MgCl2, 0.02%(w/v) BSA, 0.02 mm ATP, 8 Mm NADP, 3.5 Mg hexokinase (Boehringer-Mannheim Corp., Cat. No. 127817), 0.04 MAg glucose-6-dehydrogenase (Boehringer-Mannheim Corp., Cat. No. 127035). For fructose, 20 Ml of sample was added to 2 ml of a solution containing 0.4 Mg phosphoglucoisomerase (Boehringer-Mannheim Corp., Cat. No. 128139) in addition to the reagents used for glucose. A 10-,Ml sample was added to 2 ml of the fructose assay mixture to which was added 80 units (0.2 mg) of invertase (Sigma Chemical Co., Cat. No. 1-4104). Fluorescence was determined with a Farrand Spectrophotometer Mark IV (exciter, 345 nm; emitter, 470 nm) following a 30-min incubation at room temperature. Radioactivity in starch was assayed by counting 25 Ml of the sample by liquid scintillation following conversion to glucose. Debris was first removed by centrifugation for 5 min at 1200 rpm in a clinical centrifuge. CO2 Treatment. Following the second labeling period, paired source leaves were allowed to photosynthesize at a CO2 concentration at 340 Ml 1' (air) for 2 h followed by a period at lowered CO2. Starch accumulation was determined at 15-min intervals. As a consequence of using mixtures of N2 and air to achieve the treatments, 02 concentration was reduced, reaching approximately 5% 02 (v/v) for the 75 MAl CO2 1' mixture. CO2 compensation point, approximately 50 A 1-', was obtained by circulating gas in the closed system without addition of CO2. To determine the NCE rate and corresponding CO2 concentration at which net starch accumulation was 0, CO2 concentration was lowered from 340 to 200 Ml I' and then, in increments of 25 Ml -', to compensation point. Each level was maintained for 2 h and leaf samples removed at 1-h intervals. This study of stepwise -reduction was repeated on the leaves during the next day. Subsequently, the carbohydrate status of source leaves at 340 Md 1' and at 120 Ml I` or at compensation point was compared. Treated leaves were sampled at 25-min intervals to determine
Plant Physiol. Vol. 76, 1984
starch and sugar content. The large number of samples and the limited area of the leaf allowed only two punches to be removed at each sampling time. Processing of the disks was modified by using half the volume described above for the final extract and digestion volumes. Petiole Girdling. The effect of inhibiting export on leaf carbohydrate under lowered NCE was studied by heat killing a length of petiole 1 cm below the lamina prior to lowering CO2 concentration.
RESULTS Rates of starch accumulation were measured at various NCE rates obtained by systematically lowering the concentration of CO2 in the atmosphere around a leaf (Fig. 1). Near CO2 compensation point, changes in starch content per area of leaf resulted not only from reduced starch synthesis but also from an observed decrease in area attributed to water deficit that developed as stomatal aperture incrased at low CO2. To allow for the shrinkage, starch content was expressed on a dry weight basis (Fig. IB) as well as on an area basis (Fig. IA). The CO2 concentration range that resulted in zero starch accumulation was 115 to 120 ,Ml I-'. NCE and starch accumulation rates in air and 120 Md CO2 1-' for other sugar beet plants are presented in Table I, confirming the near-zero starch accumulation rate at this CO2 level. Carbohydrate pool changes that resulted from lowered carbon I
0.50
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0.401 0 0
0.30 0.20 0
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-0.30
I, 0.125 w 0
0
0.100
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0
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0.075
