On the Mechanism of Aging in Soybean Seeds

When seeds of soybeans (Gycuine mar Amsoy var.) or safflower were stored under high 02 concentrations, their per cent germination declined rapidly. For example, soybean seeds stored under 7.7 atmospheres 02 pressure at 25°C and 17% moisture lost afl viability within 22 days, whereas under 7.7 atmospheres N2, the per cent germination remained greater than 80%. Germination decreased continually in 02 pressures ranging from 0 to 7.7 atmospheres. To avoid contamination by microorganisms during germination tests, we found a brief treatment of the seeds with 1% NaOCl solution to be essential. This treatment caused no decrease in per cent germination when compared to seeds tested without this surface sterilization. Furthermore, in our preliminary experiments, we observed that similar 02-dependent seed death occurred in both NaOCl and untreated seeds. To measure germination, samples of at least 50 seeds were immersed in distilled H20 for 30 min to allow imbibition (first 5 min in 1% sodium NaOCl for surface sterilization) and placed on filter paper-lined, 9-cm Petri dishes (25 seeds/dish), to which 7.5 ml distilled H20 was added. Seeds in dishes were incubated in darkness at 25°C for 3 d before scoring. Seeds with emerged, intact radicles were considered to have germinated. The per cent germination of the sample was taken as a measure of its viability. All experiments have been performed at least twice and in each case the results from the different sets of experiments are in good agreement. Conclusions from each experiment are statistically valid at the 95% confidence level.For those experiments in which it was desired to raise the seed moisture above its initial level of 10%o, seeds were rinsed in 1% NaOCl solution and soaked in distilled H20 for various times (2-5 min). The moisture content of the seeds was determined by difference in weight after drying for 3 h at 130°C in a forced air oven and is expressed as per cent of fresh weight of the seeds. Drying for 3 h in our oven was observed to yield constant weight.For high-pressure treatments, seeds were contained in stainless steel chambers of a model L.G.P. Oxygen Aging Block' (Scott Testers, Inc., Providence, RI). High purity oxygen and nitrogen were obtained from Matheson (East Rutherford, NJ) and used without further purification.In experiments designed to approximate published procedures for accelerated aging (1,14,15,17,18)

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Oxygen-Dependent Aging of Seeds

Ohlrogge

Kernan²

1982

“…The initial time course of solute leakage from the imbibing cotyledon also shows a period of rapid, nonlinear leakage, followed by a linear phase beginning at about the same time that water entry becomes linear. It has been suggested that the initial rapid phases of each of these events represented a period during which the membranes were relatively disorganized, and the linear phases represented steady-state processes through the reorganized membranes (7,8,11 (6.7-8 mm). Some cotyledons were heatkilled at 100 C overnight.…”

Section: Resultsmentioning

confidence: 99%

Temperature Effects on Soybean Imbibition and Leakage

Leopold¹

1980

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As a part of an analysis of the nature of chilling injury to seeds, measurenents were made of the nitial linear rates of water entry into and solute leakage out of cotyledons of soybean at various temperatures.Arrhenlus plots were approximatel linear for water entry Into both lving and dead cotyedons, with the slpe (and activation energy) for entry Into iving celis being insipificantly higher than for dead cells, suggesting lttle effect of membrane barriers on water entry. The plots for solute leakage showed 10-fold lower leakage rates from lving than from dead tissues; a reversal of slo in the Arrhenlus plot at temperatures below 15 C reflected increasing leakage rates, interpreted as a quantitative disruptio of membrane reorganization at the temperatures associated with chilng injury.peratures between 1 and 40 C were maintained by immersing the imbibing system in a water bath.The time course of solute leakage was measured as the rise in conductance of 25 ml of water containing 10 cotyledons. Conductance was measured with a Markson ElectroMark analyzer at 5-min intervals.Rates were determined from the slope of the 10-to 40-mm periods, using calculated regression lines. The logarithms of these slope values were plotted as Arrhenius plots, and activation energies were then calculated using the equation of Stein (13): 2.3 log k= ( I where Ad is the activation energy, k' and k" are log rates for 40 and 5 C (T' and T", respectively), and R is the gas constant. RESULTSEvidence has been accumulating that chilling injury to seeds during imbibition might be attributed to disruptive effects on the reorganization of membranes as water enters the dry seed (1, 9). A quantitative means of analyzing the barrier effectiveness of membranes could utilize the initial linear rates of water entry and of solute leakage from soybean cotyledons, and their analysis through Arrhenius plots.The time course of initial water entry into dry cotyledons shows a period of rapid, nonlinear entry lasting 5-10 min, followed by a period of linear water entry lasting 30 min or more (7,11). The initial time course of solute leakage from the imbibing cotyledon also shows a period of rapid, nonlinear leakage, followed by a linear phase beginning at about the same time that water entry becomes linear. It has been suggested that the initial rapid phases of each of these events represented a period during which the membranes were relatively disorganized, and the linear phases represented steady-state processes through the reorganized membranes (7,8,11). If this interpretation is correct, linear rates of water entry and of solute leakage should reflect the effectiveness of membranes in resisting the passage of water and of solutes, respectively.The experiments reported here measure the linear rates of water entry and solute leakage of cotyledons at various temperatures to permit construction of Arrhenius plots and estimation of the energy barriers provided by the reconstituted membranes. MATERIALS AND METHODSWater entry and solute leakage were mea...

“…Alterations in the membranes of aged seeds are considered to lead to a greatly enhanced leakage of solutes during seed imbibition (2,13). This may be indicative of an inability to re-form coherent membranes during rehydration of the seed, resulting in loss of vigor and lack of germination.…”

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Absence of Lipid Oxidation during Accelerated Aging of Soybean Seeds

Priestley¹,

Leopold²

1979

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When seeds of soybean were subjected to accelerated aging, the amount of total lipid which was extracted from the whole seed increased with "age," whereas the extractable phospholipid decreased slightly. This small decline primarily reflected changes in the amounts of phosphatidylchoUne and phosphatidylethanolamine. The levels of unsaturated fatty acids in the whole seed and in the seed axis showed no decline during aging. Similarly, the fatty acids in a polar Upid extract from the whole seed showed Uttle change in unsaturation. These results suggest that oxidation of seed Upids may be unrelated to the process of seed aging.The aging of dried seeds in storage is thought to be accompanied by changes in membranes (8, 24) and nucleic acids (15). Alterations in the membranes of aged seeds are considered to lead to a greatly enhanced leakage of solutes during seed imbibition (2, 13). This may be indicative of an inability to re-form coherent membranes during rehydration of the seed, resulting in loss of vigor and lack of germination. Changes in dry or rehydrated membranes of aged seeds have been noted at the ultrastructural level (6). Lesions in nucleic acids may also be important in deterioration of seed (15), although probably less significant during the first minutes of imbibition when protein and nucleic acid synthesis is probably minimal.Several workers have argued that the alterations to seed membranes during aging may result from peroxidation (10, 24). Autooxidation of polyunsaturated fatty acids leads to free radical formation and possible peroxidative damage to lipids, proteins, and nucleic acids. Peroxidation of membrane lipids gives rise to increased permeability of artificial bilayers (23) and a decrease in membrane fluidity (5). Spencer et al. (20) noted an increased level of oxygenated fatty acids in seeds of Cichorium and Crepis during long term storage, although these workers supplied no germination data. Most studies have been performed using systems of accelerated aging, in which the symptoms of natural aging are induced over a relatively short time span by exposing the seeds to conditions of high temperature and humidity (4). Koostra and Harrington (10) applied TLC to lipids extracted from cucumber seed and suggested that any phosphorus-containing spot which remained immobile in their solvent system was probably a peroxidized phospholipid; applying this principle, they indicated that peroxidation increased severalfold during aging, peroxidized phospholipids representing over 20%o of the total phospholipids in seeds rendered nongerminable. UV spectroscopy of seed fatty acids of Protea compacta by Van Staden et al. (22) ysis can be considered only semiquantitative. Harman and Mattick (7) studied accelerated aging of pea seeds and found that the decrease in germination rate was paralleled by a pronounced decline in linoleic (C 18:2) and linolenic (C 18:3) acids whereas the saturated and monoenoic fatty acids remained unchanged. They suggested that this decline in dienoic and trienoic fatty a...