Transient Changes During Soybean Imbibition

Leakage of electrolytes, substances absorbing UV light, and enzymic activities from imbibing soybean (Glycine max IL.I Menf.) seeds were compared to determine the extent that passive diffusion and cellular rupture contribute to each. Imbibing seeds with testae removed had average Arrhenius energies of activation (5 to 25°C) of 3.0 and 15.8 kilocalones per mole, respectively, for the leakage of electrolytes and embryo malate dehydrogenase activity. Leakage of embryo enzymes from imbibing seeds was dependent on loss of testa integrity and subsequent loss of cellular membrane integrity or inability to seal preexisting membrane discontinuities. These data suggest that electrolyte leakage from imbibing seeds is primarily by passive diffusion, whereas the diffusion of intraceDlular macromolecules is primarily dependent on physiological phenomena affecting membrane integrity. Kinetic data and examination of the composition of seed leachates indicated that the leakage of substances absorbing UV light during imbibition is due to both passive diffusion of low molecular weight solutes and macromolecules released from ruptured cells.ferences one would see in the kinetics of leakage of various seed components. If the assays used for seed leakage do not correlate with one another under most experimental conditions they should not be used interchangeably, as they have in the past, to study the leakage processes.In this study, we utilized imbibing soybean seeds to examine commonly used seed leakage assays to gain insight on the contribution of passive diffusion through membranes and cellular rupture (preexisting or induced) to each measurement. Seeds with various degrees of testa integrity were used because testa integrity is a primary factor in controlling legume seed leakage and cellular damage during germination (7,11,12,21,24). Effects of temperature were studied to gain insight into the energetics of the leakage of various seed components during imbibition and effects of 4,2 were studied because of previous studies which indicate a 4iw effect on soybean seed leakage and seedling vigor (28, 29). Our findings indicate that passive diffusion of low mol wt solutes and membrane rupture-dependent diffusion of macromolecules during seed imbibition are quite different kinetically and that the contribution of each process is variable among the methods used for measuring seed leaching.

Section: Resultssupporting

confidence: 59%

“…The most common measurements of seed leakage in steep water are conductivity of electrolytes (1,3,7,14,16,19,22,24,25,27,30) and UV light absorbance at various wavelengths (5,7,17,19).…”

mentioning

confidence: 99%

Differential Leakage of Intracellular Substances from Imbibing Soybean Seeds

Duke

Kakefuda²,

Harvey³

1983

“…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.…”

Section: Resultsmentioning

confidence: 98%

Temperature Effects on Soybean Imbibition and Leakage

Leopold¹

1980

Self Cite

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...

“…The respiration of soybean particles was characterized by a burst of 02 consumption during the first few min of imbibition (6). This respiratory burst was not inhibited by KCN and was inhibited by SHAM (Fig.…”

Section: Resultsmentioning

confidence: 99%

Confounding of Alternate Respiration by Lipoxygenase Activity

Parrish¹,

Leopold²

1978

Self Cite

The initial burst of respiratory activity (Qo2) of Neither the biochemical nature nor the physiological significance of the cyanide-insensitive, alternate respiration is known, but its activity has now been demonstrated in a number of plant systems (2, 9) Alternate respiration is commonly defined or diagnosed as that portion of respiration which is insensitive to cyanide but inhibited by hydroxamic acids (2,8). This activity is thought to be due to an electron flow which does not involve Cyt oxidase (2, 10). We present data which show that initial in vivo 02 uptake by soybeans is not inhibited by KCN and is inhibited by hydroxamate; and that this apparent respiration may be due in part to the activity of lipoxygenase, an enzyme which catalyzes the dioxygenation of certain unsaturated fatty acids (5). emulsion in 10 mm phosphate buffer (pH 7.0) (11). A I mg/ml (3.56 mM) LA solution was used as substrate for the LOX assays. LOX was dissolved in 10 mm phosphate buffer (pH 7.0) and injected into the 02 electrode reaction vessel to give a reaction mixture which had 100 ,ug of LOX/ml. Assuming a mol wt of about 100,000 (1), LOX concentration was about I ,UM. MATERIALS AND METHODS RESULTS AND DISCUSSIONThe respiration of soybean particles was characterized by a burst of 02 consumption during the first few min of imbibition (6). This respiratory burst was not inhibited by KCN and was inhibited by SHAM (Fig. 1). The initial respiratory activity was also markedly inhibited by PG (Fig. 2), being about 10 times more sensitive to PG than to SHAM. If the normally alkaline KCN solution was strongly buffered (100 mM) to maintain the pH at neutrality, the small promotion of respiration seen in Figure 1 was no longer observed; and there was still no evidence of inhibition in the first few min. SHAM and PG sensitivities were not affected by buffering at pH 7.0.Suspecting that the SHAM inhibition might be related to LOX activity, we tested the cell-free enzyme's sensitivity to the hydroxamate and found (Fig. 3) that indeed it was, being 90%o inhibited by 5 mm SHAM. Autooxidation of LA (in the absence of LOX) was unaffected by SHAM. The LOX activity was not inhibited by KCN (data not shown). The enzyme was about 10 times more sensitive to PG than to SHAM, being 90%o inhibited at about 0.5 mM PG (data not shown).To investigate further the possible role of LOX in the apparent initial respiration, soybean seed particles were placed in buffered solution with or without SHAM and 02 uptake was measured with and without the addition of 50 jig/ml (0.18 mM) LA substrate (Fig. 4). In the absence of SHAM, when LA was added, there was an immediate 9-to 10-fold increase in respiratory rate and all of the 02 remaining in the solution was quickly consumed. When 5 mM SHAM was present, the initial 02 consumption was inhibited approximately 50%1b, and the addition of LA caused only a transient 2-to 3-fold increase in 02 consumption. (Added Tween 20 was without effect on the respiration rates in the presence of absence of hydroxamate.) The m...