Low Temperature Effects on Soybean (<i>Glycine max</i> [L.] Merr. cv. Wells) Mitochondrial Respiration and Several Dehydrogenases during Imbibition and Germination

Duke, Stanley H.; Schrader, L. E.; Miller, Marna Geyer

doi:10.1104/pp.60.5.716

Cited by 75 publications

(35 citation statements)

References 17 publications

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“…The reaction was initiated with 0.5 ml 5% (w/ v) soluble starch (Sigma) and terminated after 10 min by adding 0.2 ml alEaline dinitrosalicycic acid solution (2) and then placing immediately into a boiling water bath. Color was fully developed after 5 min, and tubes were removed from the bath, allowed to cooL diluted with 10 profile. For preparation of the Arrhenius plots, the routine assay was used, except that the enzyme and buffer mixture was allowed to incubate at the appropriate temperature for 10 min before initiating the assay.…”

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“…For preparation of the Arrhenius plots, the routine assay was used, except that the enzyme and buffer mixture was allowed to incubate at the appropriate temperature for 10 min before initiating the assay. Ea2 values were determined as previously outlined (10 Viscosity experiments were conducted using an Ostwald-type viscometer at 30°C, with treatments described by Hultin (12).…”

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Beta-Amylases from Alfalfa (Medicago sativa L.) Roots

Doehlert¹,

Duke²,

Anderson³

1982

Plant Physiol.

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Amylase was found in high activty (193 international units per milliam protein) in the tap root of alfalfa (Medcago satia L. cv. Sonra). The activity was separated by gel filtration chromatography into two fractions with molecular weights of 65,700 (beavy amylase) and 41,700 (light amylase). Activty staining of electropboretic gels indicated the presence of one isozyme in the heavy amylase fraction and two in the liht amylase fraction. Three amylase isozymes with electropboretic mobilties identical to those in the heavy and the light amylase fractions were the only amylases identified in crude root preparations. Both heavy and lht amylases hydrolyzed amylopectin, soluble starch, and amylose but did not hydrolyze puflulan or p-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato P-amylase, while that of bacterial a-amylase was considerably higher. The identification of maltose and -limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are ail .8-amylases. Beta-amylase (a-1,4-glucan maltohydrolase, EC 3.2.1.2) is an exoamylase that attacks the nonreducing ends of starch molecules, producing ,B-maltose and fl-limit dextrin as products (22). Betaamylases are strictly plant enzymes that have been reported in ungerminated wheat and soybean seeds (11, 23); germinating barley, rice, sorghum, and wheat seeds (3,6,7,15); sweet potato roots (1); broad bean leaves (5); and pea seedling roots (21).Amylases are of particular interest in the perennial legume alfalfa (Medicago sativa L.), which stores large quantities of starch in its fleshy tap root. This starch serves as a carbohydrate reserve that is used for winter survival and shoot regeneration following harvest of the shoot as a forage (17,19). Amylase has been reported in high activity in the tap root of several alfalfa cultivars (8), and activity staining of electrophoretically separated proteins on polyacrylamide gels has shown the presence of two or three isozymes of amylase in alfalfa tap roots (13). In this study, we describe the separation, identification, and characterization of isozymes of 8-amylase from alfalfa root tissues. MATERIALS AND METHODS Plant Material. Alfalfa (Medicago sativa L. cv. Sonora) was planted in the spring as described previously (8). In the autumn of the seeding year, plants were collected, shoots clipped to within 2.5 cm of crowns, and all secondary roots removed from tap roots. Trimmed plants were either potted and maintained in a greenhouse or washed, wrapped in wet cheesecloth and plastic, and stored at -2°C until use. Greenhouse-grown plants were inoculated with a mixture of Rhizobium meliloti strains (Nitragen Co., Milwaukee, WI) and irrigated with a nitrogen-free nutrient solution (9).Separation of f8-Amylase Isozymes. Whole tap roots were washed, chopped into small pieces with a razor blade, and then mechanically homogenized with a 'homogeniser' (Measuring and Scientific Equipment,...

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Beta-Amylases from Alfalfa (Medicago sativa L.) Roots

Doehlert¹,

Duke²,

Anderson³

1982

Plant Physiol.

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“…All dehydrogenase and dehydrogenase-linked enzyme assays were conducted spectrophotometrically measuring the reduction of NAD(P)+ or the oxidation of NAD(P)H at 340 nm with a Pye Unicam SP8-100 double-beam spectrophotometer at 30 C. Except for the assay buffer (100 mm Hepes), assays are as previously described for GDH2 and MDH (7), NADP-ICDH and G6P-DH (8), and GOT (9). Assays for AK were coupled to G6P-DH and reaction mixtures (2 ml) contained, in order of addition and in final concentration: 75 from horse heart (Sigma), reduced with ascorbate and dialyzed with 1,000 volumes H20 for 18 h at 5 C; A5w/Aw = >20), and 50 A1 enzyme preparation to a final volume of 1.0 ml.…”

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Role of the Testa in Preventing Cellular Rupture During Imbibition of Legume Seeds

Duke

Kakefuda²

1981

Plant Physiol.

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105

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Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbibition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose--phosphate dehydrogenase) or and that, with removal of the testa from seeds, the "leakage phenomenon" is enhanced (14,32). The amount of leakage during imbibition has been shown to correlate negatively with viability in studies with seeds of soybean (6, 39), pea (14,24), bean (17), and peanut (1) and has suggested to some that the leaked substances may, in some way, decrease viability. Another study has shown that removal of the testa of pea seeds results in death of the outer layers of cotyledonary cells during imbibition (25). The question arises: does the testa protect against leakage or is the leakage only a symptom of a fundamental dysfunction which can occur in imbibition?Two hypotheses have been promoted to explain the mechanism(s) ofleakage of solutes during the imbibition ofseeds. Larson (14) has suggested that cell membranes are ruptured during the initial phases of imbibition. Simon (31, 33) has proposed that the membranes of dry seeds are formed into hexagonal plates with pores formed in the areas of the phospholipid heads through which low-molecular weight solutes can leak from cells by passive diffusion during initial stages of membrane hydration (e.g. before phospholipids form typical bilayer membranes). Recently Powell and Matthews (25) have suggested that, in peas, cellular rupture and leakage through membranes may both occur when the testae are removed from seeds. To date, there has been a paucity of data that any macromolecules could move through the cell membrane during imbibition. Here, we have examined the leakage of imbibing seeds with and without testae for the presence of cytoplasmic, organelle, and organelle marker enzymes which would not pass through small membrane pores but which could only pass through very large membrane discontinuities or which would be the result of membrane rupture. In this way, we have tested both of the aforementioned hypotheses in a more definitive manner than has been hitherto reported.In the development of the legume seed, the testa appears to function in interconverting amino acids and sugars supplied by the phloem to the developing embryo (19,35,36) and in preventing injury by differentiating into a sclerified integument as the embryo matures (27). It has also been proposed that the testa protects seeds against "leakage" of intracellular substances during imbibition (32). This function has been suggested to be of great importance in the initial stages of germination of legume seeds in that many substances which leak from seeds may offer a substrate for potential pathogens (32). Past studies have demonstrated that electrolytes, sugars, amino acids, organic acids, and proteins are released from seeds during imbibition (1,6, 14,18,23,29,33,34) Mammoth Virgi...

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“…Further, glutamate dehydrogenase was also inhibited, restricting the flow of aketoglutarate from the cycle (Duke et al, 1977). Buescher (1975) observed an increase in citrate and a decrease in malate in tomato pericarp tissue in response to low temperature, which would be consistent with the effects of chilling on Krebs cycle enzymes.…”

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confidence: 48%

“…Buescher (1975) observed an increase in citrate and a decrease in malate in tomato pericarp tissue in response to low temperature, which would be consistent with the effects of chilling on Krebs cycle enzymes. Two enzymes of glycolysis, phosphofructokinase (Graham and Patterson, 1982) and glyceraldehyde-3-phosphate dehydrogenase (Guy, 1990) and one enzyme of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase (Duke et al, 1977) are also known to be inactivated by chilling. Along with the reduction in the terminal oxidase, all of these changes illustrate how respiration is greatly reduced by chilling, and how greater susceptibility of some respiratory components to chilling relative to others can lead to dramatic changes in the pools of various metabolites.…”

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Molecular and biochemical investigation of the mechanisms of acclimation to chilling stress in maize seedlings

Anderson

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Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Mitochondrial Respiration and Several Dehydrogenases during Imbibition and Germination

Cited by 75 publications

References 17 publications

Beta-Amylases from Alfalfa (Medicago sativa L.) Roots

Beta-Amylases from Alfalfa (Medicago sativa L.) Roots

Role of the Testa in Preventing Cellular Rupture During Imbibition of Legume Seeds

Molecular and biochemical investigation of the mechanisms of acclimation to chilling stress in maize seedlings

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