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Characteristics of Carbohydrate Metabolism in Sweet Corn (Sugary-I) Endosperms

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Characteristics of Carbohydrate Metabolism in Sweet Corn (Sugary-I) Endosperms Reprints from Vol. 118(5), September 1993 Journal of the American Society for Horticultural Science 1071 J. AMER. Soc. HaRT. SCI. 118(5):661-666. 1993. Characteristics of Carbohydrate Metabolism in Sweet Corn (sugary-I) Endosperms Douglas C. Doehlertl and Tsung Min Kuo U.S. Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Phytoproducts Research Unit, 1815 North University Street, Peoria, IL 61604 John A. Juvik Department ofHorticulture, University ofIllinois, 1201 West Gregory Avenue, Urbana, IL 61801 Eric P. Beers and Stanley H. Duke Department ofAgronomy, University ofWisconsin, 1575 Linden Drive, Madison, WI 53706 Additional index words. Zea mays, phytoglycogen, gene dosage Abstract. Metabolic characteristics ofdeveloping sugary-! maize (Zea mays L.) endosperms were investigated. In the later stages ofdevelopment (>30 days postpollination), sugary-! kernels maintained higher levels ofmany enzyme activities and retained more moisture than normal kernels. Higher enzyme activities were attributed to moisture retention and were not associated with any increase in dry weight accumulation. Ofenzyme activities measured at 20 days postpollination, that ofADP-glucose pyrophosphorylase was higher in sugary-! kernels than in normal, whereas total amylase, a-amylase, and pullulanase activities were lower. Experiments testing the effects of zero, one, two, and three doses of the sugary-! gene in OH43 endosperms indicated that the sugary-! phenotype was not expressed until three doses ofthe sugary-! gene were present. Decreased activities of amylases, but not of pullulanase, were attributed to an interference in detection by phytoglycogen. Increased ADP-glucose pyrophosphorylase activity is attributed to a response by the maize endosperm cells to increased sucrose concentrations. The sugary-l genotype ofmaize (Zea mays), commonly grown maize kernel. Ourresults provide some additional insights into the as sweet corn, accumulates more sugars in the endospenn than nature ofthe sugary-1 mutation and contribute infonnation on the nonnal starchy maize. The principle difference between nonnal functioning of the nonnal maize kernel. and sugary-l maize is that sugary-l endospenns accumulate the highly branched, water soluble form of starch known as Materials and Methods phytoglycogen (Morris and Morris, 1939). There are two hypoth­ eses concerning the origin of phytoglycogen. The first suggests Plantmaterial. Inbred and hybrid maize lines were grown in the that sugary-l maize kernels lack sufficient starch debranching field in Peoria, il1., during 198~88. Nonnal and sugary-1 isolines enzyme (Erlanger, 1957). This hypothesis suggests that starch of OH43 were obtained from the Maize Genetics Cooperative of biosynthesis involves the synthesis of a highly branched intenne­ the Unlv. of illinois, Agronomy Dept., Urbana. onnal and diate fonn ofstarch which is subsequently debranched to fonn the sugary-1 isolines ofW64A were obtained from L. Darrah, (Univ. amylose and amylopectin found in nonnal maize starch granules. of Missouri, Columbia). Plants grown in the field were hand­ Evidence supporting this hypothesis was provided by Pan and pollinated and harvested at designated time intervals. Samples Nelson (1984), who demonstrated significantly reduced pullulanase were taken in a randomized design with three or four replications. activity (one fonn of starch debranching enzyme found in devel­ For enzyme extractions and carbohydrate analyses, immature oping maize kernels) in sugary-1 kernels that appeared to depend kernels were stripped from harvested ears, dissected, and about on the sugary-l gene dosage. thirty endospenns were lyophilyzed and stored at-80 C until used. The second hypothesis suggests that the sugary-1 mutation Whole kernels also were stripped from freshly harvested ears and affects a starch-branching enzyme, which results in the more frozen at-80C until used. Kernel dry weights and moisture content highly branched phytoglycogen (Boyer and Preiss, 1978). The were detennined after drying in a forced air oven at 60C for 72 h. sugary-1 mutation has many additional effects on maize kernel Enzyme assays and carbohydrate analysis. Extraction and development. Amyloplasts fail to fonn birefringent starch gran­ assays of enzymes were perfonned as described by Doehlert et al. ules and instead accumulate phytoglycogen (Boyer et al., 1977). In (1988) and Doehlert and Kuo (1990). Enzymes were extracted by comparison to nonnal kernels, sugary-l kernels accumulate less homogenizing 0.2 g powdered lyophilized tissue in 4 ml extraction , dry weight (Andrew et a1., 1944; Tsai et al., 1978), retain kernel buffer containing (in mM) 50 HEPES (pH 7.2), 5 MgCI2 and 5 moisture longer (Andrew et al., 1944), have thinner pericarp dithiothreito1. Particulates were settled by centrifugation of the (Andrew et al., 1944) and contain altered storage protein (Tsai et homogenate at 10,000x g for 15 min. al., 1978). The enzymes sucrose synthase (EC 2.4.1.13), pyrophosphate: In this study, we have investigated developmental and dosage D-fructose 6-phosphate, l-phosphotransferase (EC 2.7.1.90; PFP), effects of the sugary-1 gene on carbohydrate metabolism in the UDP-Glc pyrophosphorylase (EC 2.7.7.9), ADP-Glc pyrophos- Received for publication 1 Sept. 1992. Accepted for publication 12 Jan. 1993. The cost ofpublishing this paper was defrayed in part by the payment ofpage charges. Abbreviations: ADP-Glc, adenosine diphosphate glucose; DPP, days postpollina­ Under postal regulations, this paper therefore must be hereby marked advertise­ tion; HEPES, N-2-hydroxyethylpiperazine- '-2-ethanesulfonic acid; PFK, phos­ ment solely to indicate this fact. phofructokinase; PFP, pyrophosphate: fructose-6-phosphate, l-phosphotransferase; ITo whom reprints requests should be addressed. UDP-Glc, uridine diphosphate glucose. J. Amer. Soc. Hart. Sci. 118(5):661-666. 1993. 661 phorylase (EC 2.7.7.27), aldolase (EC 4.1.2.13), AD-dependent pooled and brought up to 50 ml, and glucose concentrations were sorbitol dehydrogenase (EC 1.1.1.14), phosphoglucoisomerase determined with the glucose oxidase method (Gascon and Lampen, (EC 5.3.1.9), phosphoglucomutase (EC 2.7.5.1), phosphofruc­ 1968). Soluble sugars, phytoglycogen, and starch in mature sug­ tokinase(EC2.7.1.11), glucokinase (EC2.7.1.11), andfructokinase ary-I gene dosage series kernels were fractionated by the proce­ (EC 2.7.1.4) were assayed by continuous spectrophotometric dure described by Dickinson et al. (983) and quantitated by the assays where the reaction product was coupled with either AD phenol sulfuric acid method (Hodge and Hofrieter, 1962). reduction or ADH oxidation and monitored by A as described 340 Phytoglycogen was purified from OH43 sugary-1 kernels by the by Doehlert (990) and Doehlert et al. (988). method of Schoch (957). Pullulanase (EC 3.2.1.41) was assayed by the procedure of Student's t tests and analyses of variance were performed with Doehlert and Knutson (991). Assays contained 50 ITlM acetate­ the ABSTAT (Anderson Bell, Parker, Colo.) computer statistics , NaOH (pH 5.0), 5 mM MgC1 2 and 2.5% pullulan (w/v). Samples package. Least significant differences were calculated by the were taken at 0,10,20, and 30 min. Reducing power ofthe sample method described by Steel and Tome (960). was determined with dinitrosalicylic acid solution (Bernfield, 1951), and compared with a maltotriose standard curve. An addi­ Results and Discussion tional blank was used containing enzyme extract but no pullulan to correct for the contribution of hydrolyzed phytoglycogen to the During kernel development, sugary-1 kernels had significantly increase in reducing power. Total amylase activity was assayed by higher fresh weight than normal kernels (P> 0.01; Table 1) from measuring the increase of reducing power in a solution of soluble 10 through 35 DPP, and had significantly lower dry weight from starch. Assays contained 50 mM citrate (pH 6.0) and 5% soluble 20 DPP to maturity (P > 0.05, Table 1). Likewise, sugary-1 kernels starch (w/v). Assays were terminated after 10 min, and the reduc­ had higher moisture contents at20, 25, 30, and 35 DPPthannormal ing power was measured with dinitrosalicylic acid solution (P > 0.05; Table 1). Normal mature (9% moisture) kernels had (Bernfield, 1951). The a-amylase activity was assayed by the significantly higher fresh and dry weights than mature sugary-1 starch azure method (Doehlert and Duke, 1983). Assays contained kernels. Some of the differences in kernel dry weights can be (in mM) 50 citrate buffer (pH 6.0), 5 dithiothreitol, 2 CaC1 , and 20 2 attributed to losses in sugary-1 kernel dry weight during the dry­ g starch azure/liter. Interference by ~-amylase was eliminated by down period ofkernel development (after 30 DPP). The sugary-1 adding 1000 units sweet potato ~-amylase/m1. The solubilized kernels lost about 8% of their dry weight during this period, blue pigment indicative ofa-amylase activity was measured at 595 whereas normal kernels lost only 2%. A comparison of means by nm and was compared with that released by known activities of a Student ttest indicated that there was a significant decrease in dry bacterial a-amylase in the presence of 1000 units/ml sweet potato weight in the sugary-1 genotype (P > 0.01), whereas the decrease ~-amylase. Doehlert and Knutson (991) indicated that starch in dry weight in the normal genotype was not significant. Similar debranching enzyme activities had no effect on the starch azure decreases
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