Biochemical Properties of Mitochondrial Membrane from Dry Pea Seeds and Changes in the Properties during Imbibition

The free amino acid concentrations in cotyledons and axes of soybean (Glycine max IL.I Merr. cv. Wells) seedlings were determined by automated single column analysis after germiation at 10 and 23 C. After 5 days germination at 10 C, glutamate and aspartate were in high concentration in both cotyledons and axes (38 and 24% of total free amino acids recovered, respectively), whereas the concentrations of their amide derivatives, asparagine and glutamine, were iow in cotyledons (4.4%) and high in axes (21%). In contrast, after 5 days germination at 23 C, asparagine and glutamine accounted for 22 and 45% of total free amino acids in cotyledons and axes respectively, and aspartate and glutamate concentrations were low. The activities of glutamine synthetase and asparagine synthetase were considerably lower in tissues from the 10 C treatment than those from the 23 C treatment.Aspartate and glutamate concentrations were nearly equal in all but one sample. Both glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were much higher in axis tissues at 23 C as compared to 10 C. Arrhenius plots of axis glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were biphasic and triphasic, respectively, with energies of activation for both increasing with low temperature. Energies of activation were identical for glutamate oxaloacetate transaminase from 10 and 23 C treatments but much higher for glutamate debydrogenase from 23 C-treated axes. This indicates a difference in enzyme complement for glutamate dehydrogenase with the two treatments.Hydrolysis of free amino acid sample (basic fraction) aliquots showed large quantities of peptides in 23 C-treated axes at 2 days, while few or no peptides were found in the 10 C treatment. Amino acid residues most prevalent in peptides were aspartate, threonine, serine, glutamate, and glycine.In legume germination the proportions of amino acids in storage proteins are not reflected by the proportions of free amino acids which occur during storage protein mobilization of such species as Glycine max (5,15). This is largely due to interconversions of amino acids released from storage proteins to produce both new nonprotein and protein amino acids (2).Slower soybean germination (as measured by radicle emergence from the testa) at low temperature appears to be due in part to high E.3 values at suboptimal germination temperatures for the enzymes involved in energy transduction (10). Inasmuch as amino acid interconversions are in many cases energy-requiring processes, we decided to determine the effects of low temperature on free amino acid pools in soybean seedlings. Also, because we found high levels of aspartate, glutamate, asparagine, and glutamine, we surveyed several enzymes which are considered primary in the metabolism of these amino acids (GDH, GS, AsnS, and GOT). The activities of these enzymes from soybean seedlings grown at low and optimal temperatures appear to be responsible for many of the observed differences in free amino acid pools...

Section: Methodscontrasting

confidence: 43%

Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Free Amino Acid Pools during Germination

Duke¹,

Schrader²,

Miller³

et al. 1978

“…The amount of GDH activity recovered from particulate (mitochondrial) fractions was 66.7 ± 8.8% and 36.1 ± 5.1% of the total activity for axes and cotyledons, respectively, after 48 hr at 23 C. In general, preparations increased in the percentage of mitochondrial dehydrogenases (GDH, MDH, and NADP-ICDH) recovered in the mitochondrial pellet (20,000g) over the course of the experiment (data not shown). This was true for both treatments and may have been due to increased mitochondrial integrity as has been shown to occur with imbibition in legumes (33). All GDH should have been mitochondrial in origin (7,35) at this stage of development with any recovery of the enzyme in the supernatant due to mitochondrial disintegration.…”

mentioning

confidence: 60%

“…Studies with legumes have shown that during imbibition and germination rapid changes occur in mitochondrial respiration (24,33). Dehydrogenases are of particular importance in cold acclimation due to their production of reduced nucleotides necessary for energy transduction (1,19 All dehydrogenases were assayed spectrophotometrically utilizing a Gilford 2000 spectrophotometer equipped with a H20-jacketed cuvette chamber for temperature control.…”

mentioning

confidence: 99%

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

Duke

Schrader²,

Miller³

1977

The influence of low temperature on soybean (Glycine max [L.] Meff. cv. Wells) energy transduction via mitochondrial respiration and dehydrogenases was investigated in this study during imbibition and germination. Mitochondria were isolated from embryonic axes of seeds treated at 10 and 23 C (control) by submergence in H20 for 6 hours and maintenance for an additional 42 hours in a moist environment.Arrhenius plots of initial respiration rates revealed that those from cold-treated axes had respiratory control (RC) ratios of near 1.0 above an inflection in the plot at 8 C. Arrhenius plots of control axes mitochondrial respiration showed RC rtios of 2.8 above and 5.0 below an inflection temperature of 12.5 C. Energies of activation for mitochondrial respiration between 20 and 30 C for the cold and control treatments were 7.8 and 15.6 kcal/mole, respectively. These data indicate possible differences in mitochondria membranes, degree of mitochondrial integrity, and mitochondrial enzyme complement between the two treatments.Gluhtmate dehydrogenase (GDH), malate dehydrogenase (MDH), adcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6P-DH), and NADP-isocitrate dehydrogenase (NADP-ICDH) were sayed from whole seeds and axes (after germination) during the 48 bours of temperature treatments. Activity of these dehydrogenases decased during the first 6 hours with the exception of MDH. After germination at 23 C (48 hours) all five debydrogenases increased in activity. Arrhenius plots of cotyledon debydrogenase activities indicated that one inflection temperature between 6 and 18 C was present for each enzyme assayed. Differences were seen in Arrhenius plots of axes dehydrogenase activities with the two temperature treatments in the cases of GDH and MDH from mitochondrial pellets and with differences in enzyme extraction media. These data suggest that the temperature treatments yield differences in mitochondrial enzyme complement. There were no detectable inflection temperatures for the activities of G6P-DH and ADH extracted from axes. Arrhenius plots of NADP-ICDH activity indicated extreme cold sensitivity. The dopes of the plots for axes NADP-ICDH were very similar to those for mitochondrial respiration (23 C trtment) suggesting that this enzyme may limit mitochondrial respiration at low temperature in soybean tissues grown at moderate temperatures.

“…Moreover, pelleting ofthe early mitochondria required a higher centrifugal force than that of mature mitochondria. A comparably wide distribution of Cyt oxidase in sucrose gradients has also been demonstrated after isolation of early mitochondria from several seed species (15,18,20). Since in these studies no malate dehydrogenase could be liberated from the dispersed zones of Cyt oxidase activity, the latter were taken to represent the remnants of fragile mitochondria that had lost their matrix.…”

mentioning

confidence: 99%

Isolation-Inflicted Injury to Mitochondria from Fresh Pollen Gradually Overcome by an Active Strengthening during Germination

Hoekstra

Roekel²

1983

Activities of segments of the electron transport pathway of mitochondria isolated from pollen of Typha latifolia L during the course of germination in vitro were compared with those of mitochondria in intact grains. For this purpose, suitable inhibitors and artificial substrates were selected for their ability to penetrate through the exine, intine, and plasmalemma. In contrast to their counterparts in vivo, mitochondria isolated during the initial stages of germination exhibited low rates of electron transport, resulting from loss of NAD and displacement of cytochrome c from its site of action. The phosphorylative capacity was also impaired. Great caution must be exercised therefore, before interpreting results obtained with isolated mitochondria.The gradually acquired resistance of mitochondria to injury during isolation as germination proceeds was shown to depend on an energyrequiring process and not solely on a rearrangement at the membrane level, or imbibitional differences. Dc novo syntheses of proteins or fatty acids were not required for the strengthening of mitochondria since cycloheximide, chloramphenicol, and cerulenin did not prevent this change. The nature of the energy-requiring process remains obscure. It is probable that strengthening of mitochondrial membranes during seed germination has been misinterpreted due to similar effects of isolational injury.