Superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination.

The total rate of mitochondrial O2- production in the presence of NADH as substrate increased from 200 to 1340 pmol/min per axis between 2 and 30 h of imbibition. The activities of the enzymes involved in hydroperoxide metabolism, e.g., superoxide dismutase, catalase, peroxidase and glutathione and ascorbate peroxidases, markedly changed during the germination of soybean embryonic axes. Superoxide dismutase was the enzymatic activity affected the most during the initial stages of germination. Intracellular O2- steady-state concentration, calculated from the rate of O2- production and superoxide dismutase activity, showed a 2-fold increase from 2 x 10(-8) M to 4 x 10(-8) M in germination phase I, declined in phase II to 2 x 10(-8) M and remained constant over the rest of the incubation period. The reaction of H2O2 and luminol catalyzed by Co2+ was utilized to measure H2O2 diffused out of the soybean axes after 5 to 10 min of incubation. The catalase-sensitive luminol emission of diffusates prepared from axes previously imbibed from 2 to 30 h corresponded to a H2O2 intracellular steady-state concentration in the range of 0.3 to 0.9 microM. The activity of metal-containing antioxidant enzymes was determined in the extracellular fluid. Cell wall peroxidase activity increased from 10 to 300 mumol/min per mg protein and appears as a potentially important pathway for H2O2 utilization. Hydrogen peroxide metabolism in soybean embryonic axes during early inhibition appears to have the following main features: (a) mitochondrial membranes are the most important source of cytosolic O2- and H2O2; (b) H2O2 is regulated at a steady-state concentration of 0.3-0.9 microM; (c) catalase is the main enzyme in terms of H2O2 utilization; (d) H2O2 exo-diffusion is quantitatively important destiny of intracellular H2O2; and (e) extracellular peroxidase located at the cell wall affords an enzymatic system able to use diffused H2O2.     

Purine nucleotide metabolism of germinating soybean embryonic axes

Isolated soybean (Glycine max L. cv. Kent) embyronic axes metabolized [¹⁴C]glycine to ATP within the 1 hour of imbibition. Radioactivity was not detected in GTP until the 3rd hour. Throughout most of the first 24 hours of germination about 10 to 26 times as much label from [¹⁴C]glycine appears in ATP as GTP. About five times as much [¹⁴C]hypoxanthine and [¹⁴C]inosine was converted into GTP as into ATP in embryonic axes. Two independent pools of IMP appear to be used in purine nucleotide synthesis of soybean axes.     

Adenylate metabolism of embryonic axes from deteriorated soybean seeds

RNA and protein syntheses in axes excised from dry soybean (Glycine max L.) seeds at different levels of deterioration were assayed. Low rates of protein synthesis in slightly deteriorated seeds were not due to losses in ribosomal or soluble fraction activities. However, the lowered rates of RNA and protein syntheses of deteriorated seeds were associated with reduced ATP content of the tissues. Adenine and adenosine conversions to ATP were reduced in deteriorated axes, and these reductions were reflected in reduced incorporation of these compounds into RNA.     

Shotgun proteomic analysis of soybean embryonic axes during germination under salt stress

Seed imbibition and radicle emergence are generally less affected by salinity in soybean than in other crop plants. In order to unveil the mechanisms underlying this remarkable salt tolerance of soybean at seed germination, a comparative label‐free shotgun proteomic analysis of embryonic axes exposed to salinity during germination sensu stricto (GSS) was conducted. The results revealed that the application of 100 and 200 mmol/L NaCl stress was accompanied by significant changes (>2‐fold, P<0.05) of 97 and 75 proteins, respectively. Most of these salt‐responsive proteins (70%) were classified into three major functional categories: disease/defense response, protein destination and storage and primary metabolism. The involvement of these proteins in salt tolerance of soybean was discussed, and some of them were suggested to be potential salt‐tolerant proteins. Furthermore, our results suggest that the cross‐protection against aldehydes, oxidative as well as osmotic stress, is the major adaptive response to salinity in soybean.     

Germination of soybean embryonic axes: nucleotide sugar metabolism and initiation of growth

UDP-Sugars comprise the dominant class of nucleotide sugars in isolated soybean axes during early germination. While "dry" axes contain 1 nanomole per axis of UDP-sugars, further synthesis is initiated upon imbibition such that the concentration of total UDP-sugars reaches 8 nanomoles per axis or roughly 1 millimolar after 10 hours, when the axes begin to elongate. The GDP-sugars are essentially absent before imbibition, accumulate rapidly for 90 min to 173 picomoles per axis, then decrease somewhat, reattaining the earlier peak level shortly before growth begins. Meanwhile, the level of ADP-sugars is unchanged. These data indicate that the 10-hour lag period preceding axis growth does not result from a diminished ability to synthesize a major category of nucleotide sugars.Relative rates of synthesis of individual UDP- and GDP-sugars were determined by incorporation of [(3)H]uridine or [(3)H]guanosine. The distribution of label in the different classes of UDP-sugars and in the single class of GDP-sugar was quantitatively similar when analyzed before, at the onset, or during early growth. It therefore seems unlikely that synthesis of a key nucleotide sugar controls the initiation of growth.The possible relevance of nucleotide sugars to growth is discussed and new methods for enzymic analysis of picomole levels of nucleotide sugars are described.     

Oxygen-dependent increase of antioxidants in soybean embryonic axes

Oxygen, although essential for the survival of aerobic organisms leads to generation of toxic species. The effect of oxygen on enzymatic and non-enzymatic antioxidants was determined to evaluate response to oxidative stress in soybean axes. Soybean seeds were incubated over nutrient solution-saturated filter paper. Different oxygen concentrations in the incubation atmosphere were maintained by gassing either N₂ (40% O₂), air (20% O₂), a commercial mix 40% O₂ + 60% N₂ (40% O₂) or O₂ (100% O₂) in closed plastic chambers. Oxidative stress was assessed by the oxidation of 2′,7′-dichlorofluorescein diacetate. The activities of antioxidant enzymes were determined spectrophotometrically. α-Tocopherol and ubiquinol-10 contents were measured by HPLC. The weight of axes was 13 ± 1 and 27 ± 3 mg/axis in the absence and presence of 20% oxygen, respectively. 2′,7′-Dichlorofluorescein diacetate oxidation was increased from 14 ± 2 to 66 ± 5 AU/min/mg FW by supplementation of 20% oxygen. Total glutathione content was 22 ± 6 and 33 ± 6 nmol/axis in axes grown in absence of oxygen and air, respectively. Ubiquinol-10 content was not affected by oxygen. α-Tocopherol content decreased from 384 ± 94 to 14 ± 3 pmol/axis in the absence or presence of 100% oxygen, respectively. The activities of antioxidant enzymes increased in axes exposed to oxygen. Our data suggest that exposure of soybean axes to oxygen leads to oxidative stress but damage by oxygen intermediates was limited by increases in the activity of both, antioxidant substances (i.e. glutathione) and antioxidant enzymes.     

Maturation and germination of Phaseolus vulgaris embryonic axes in culture

In this study of embryo development in Phaseolus vulgaris L., we found that immature embryonic axes placed in culture show a growth lag before germinating. The length of this lag phase varies according to axis age at excision, but is not affected by transfer to fresh medium, alteration of sucrose concentration between 0.5 and 2%, or whether the culture medium is liquid or agar-solidified. The lag phase was shortened by both actinomycin D and cordycepin treatment, and by treatment with 10^-5 to 10^-6 M benzyladenine. The effect of abscisic acid (ABA) varied with concentration: below a certain level, it had no effect on the lag phase, but above that level it inhibited, germination. This threshold concentration was 10^-7 M for 20-d-old axes but increased to 10^-5 M by the time the axes were 32 to 34 d old. To determine whether the axes were continuing their embryonic development during the lag phase, we tested them for desiccation-tolerance and for synthesis of phaseolin, a seed storage protein which is specific for embryos of P. vulgaris. The ability to germinate after rapid desiccation was acquired by axes at 26 d past anthesis; when axes younger than this were placed in culture, they developed desiccation-tolerance during the lag phase of growth, indicating that they were continuing embryonic maturation. Phaseolin was present in isolated axes, although at lower levels than in cotyledons. It accumulated during axis development in parallel with total protein, staying at about 1% of total protein content. When isolated immature axes were pulsed with ³H-or ¹⁴C-amino acids, they incorporated label into phaseolin, shown by precipitation with anti-phaseolin antibody. Isolated axes from mature seeds, however, did not synthesize detectable amounts of phaseolin. Immature axes cultured in vitro for a period of one to several days continued synthesizing phaseolin until the day prior to visible germination. Treatment of cultured axes with ABA increased the amount of precursor amino acids incorporated into protein, but had a small or no effect on the relative proportion of phaseolin synthesized. We conclude that P. vulgaris axes in culture continue to develop embryonically for a period of time which seems to be under intrinisc control by the axis. This contrasts with precocious germanation, a pattern of embryo behavior seen in many other species. When such embryos are excised from seeds while immature and placed in culture, they switch promptly from embryo development into germination. If ABA or water stress is responsible for preventing precocious germination, it may be that a high level of ABA is maintained or synthesized internally by embryonic axes of Phaseolus, while in other embryos the maternal environment supplies ABA and/or causes water stress.Abbreviations ABA Abscisic acid - BA benzyladenine     

Insulin-stimulated ribosomal protein synthesis in maize embryonic axes during germination

Addition of insulin to maize seed ( Zea mays L. cv. Chalqueño) was found to accelerate germination and seedling growth. Insulin-stimulated maize axes showed enhancement of 35 S-methionine incorporation into ribosomal proteins (rp) and mobilization of S₆ rp mRNA into polysomes. Increase in S₆ rp phosphorylation of the small ribosomal subunit (40S) was observed in 32 P-orthophosphate pulse-labeled experiments when maize axes were stimulated by insulin. Application of either wortmannin or rapamycin, inhibitors of protein kinases of the insulin transduction pathway, abolished the insulin stimulatory effect on S₆ rp phosphorylation and on ribosomal protein synthesis. The above data are interpreted as an indication of the existence of an insulin-stimulated signal transduction pathway in maize tissues that is involved in the regulation of translation.     

RNA synthesis in germinating embryonic axes of soybean and wheat

The rate of synthesis of RNA during early germination of wheat and soybean embryos was investigated by ascertaining the incorporation of radioactive uridine into RNA. In wheat embryos, where the lag period preceding rapid growth is 5.5 hours, there is a 2-fold increase in RNA synthesis between 1.5 and 5.5 hours, with half of the increase occurring by 3.5 hours. In soybean axes, where the lag period is 9.5 hours, the increased rate of RNA synthesis is 5.5-fold between 1.5 and 9.5 hours, with three fourths of this increase occurring after 4 hours.     

Ethanolamine incorporation into membranes of pea embryonic axes during germination

Changes in the ethanolamine pool of the embryonic axes of pea seeds exposed to different temperatures during imbibition and germination were followed. The ethanolamine pool decreased except during imbibition at 25°. Label from ethanolamine was incorporated almost entirely into phosphatidylethanolamine with incorporation into phosphatidylcholine being observed only after imbibition and germination at 25°. The incorporation of ethanolamine was apparently less sensitive to temperature than that of choline and glycerol, previously reported. Preliminary results also show an effect of the imbibition temperature on some of the membrane proteins, but most did not seem to be affected.     

Gibberellin-like activity in cotyledons and embryonic axes during pea seed germination

Endogenous gibberellin-like activity was determined in dry pea seeds (Pisum sativum cv. Bördi), in cotyledons and axes of germinating pea seeds and also in excised cotyledons and axes. During the first two days of pea seed germination, neither the embryonic axes nor the cotyledons show a mutual influence on gibberellin activity, but this appears after 72–96 h of germination. The gibberellin-like activity m cotyledons and axes of germinating seeds increased during the same period, but it decreased in isolated axes and excised cotyledons.     

Hydrogen peroxide release from bacterial spores during germination

The release of free H₂O₂ from spores of Clostridium perfringens and Bacillus megaterium during germination has been demonstrated using the scopoletin fluorescence assay. Scopoletin oxidation was markedly inhibited when exogenous catalase was added, and was also influenced by the concentration of spores. H₂O₂ release into the germination medium was observed to parallel the O₂ consumption during germination, suggesting that the H₂O₂ may arise from certain O₂-dependent metabolism associated with initiation of spore germination.     

Sugar metabolism in germinating soybean seeds: evidence for the sorbitol pathway in soybean axes

Characterization of sugar content and enzyme activity in germinating soybean (Glycine max L. Merrell) seeds led to the discovery of sorbitol accumulating in the axes during germination. The identity of sorbitol was confirmed by relative retention times on high-performance liquid chromatography and gas liquid chromatography and by mass spectra identical with authentic sorbitol. Accumulation of sorbitol in the axes started on day 1 of germination as sucrose decreased and glucose and fructose increased. Sucrose also decreased in the cotyledons, but there was no accumulation of sorbitol, glucose, or fructose. Accumulation of sorbitol and hexoses was highly correlated with increased invertase activity in the axes, but not with sucrose synthase and sucrose phosphate synthase activities. Sucrose synthase activity was relatively high in the axes, whereas the activity of sucrose phosphate synthase was relatively high in the cotyledons. Ketose reductase and aldose reductase were detected in germinating soybean axes, but not in cotyledons. Fructokinase and glucokinase were present in both axes and cotyledons. The data suggest a sorbitol pathway functioning in germinating soybean axes, which allows for the interconversion of glucose and fructose with sorbitol as an intermediate.     

Hydrogen peroxide spraying alleviates drought stress in soybean plants

To ascertain the effect of exogenously applied hydrogen peroxide (H₂O₂) on drought stress, we examined whether the spraying of soybean leaves with H₂O₂ would alleviate the symptoms of drought stress. Pre-treatment by spraying leaves with H₂O₂ delayed foliar wilting caused by drought stress compared to leaves sprayed with distilled water (DW). Additionally, the relative water content of drought-stressed leaves pre-treated with H₂O₂ was higher than that of leaves pre-treated with DW. Therefore, we analyzed the effect of H₂O₂ spraying on photosynthetic parameters and on the biosynthesis of oligosaccharides related to water retention in leaves during drought stress. Under conditions of drought stress, the net photosynthetic rate and stomatal conductance of leaves pre-treated with H₂O₂ were higher than those of leaves pre-treated with DW. In contrast to DW spraying, H₂O₂ spraying immediately caused an increase in the mRNA levels of d-myo-inositol 3-phosphate synthase 2 (GmMIPS2) and galactinol synthase (GolS), which encode key enzymes for the biosynthesis of oligosaccharides known to help plants tolerate drought stress. In addition, the levels of myo-inositol and galactinol were higher in H₂O₂-treated leaves than in DW-treated leaves. These results indicated that H₂O₂ spraying enabled the soybean plant to avoid drought stress through the maintenance of leaf water content, and that this water retention was caused by the promotion of oligosaccharide biosynthesis rather than by rapid stomatal closure.     

Effect of temperature of imbibition on phospholipid metabolism in pea embryonic axes

Pea seeds were imbibed in radioactive choline and then germinated. Treatments were either at 5° or 25° and the seeds were imbibed for 5 hr at one temperature and then transferred to the other. [$\text{Me}$-¹⁴C]Choline incorporation into phosphatidyl choline in the ER and the plasma membrane obtained from the embryonic axes after germination was measured. Seeds kept constantly at 25° had a very rapid initial incorporation of choline followed by a loss of label. Seeds kept at 5° had a very much lower rate of incorporation. However, seeds transferred from 5 to 25° behaved for at least 48 hr as if continuously kept at 5°, while in seeds transferred from 25 to 5° incorporation stopped after 15 hr. The seeds apparently respond to transient exposure to temperature by a changed metabolism of phospholipid. Data are also given for the choline content of the seeds under the different treatments and for the changes in total phospholipid.     

Nitric oxide decreases superoxide anion generation by microsomes from soybean embryonic axes

Experiments were carried out to evaluate the effects of exposure to nitric oxide on the ability by NADPH‐dependent microsomal electron transfer to generate oxygen radicals. Such interactions could play a role in the potential antioxidant action of nitric oxide (NO). Isolated microsomes from soybean ( Glycine max [L.] Merr. cv. Hood) embryonic axes were exposed to an exogenously added source of nitric oxide (NO) (S‐nitrosoglutathione + dithiothreitol). The O₂⁻ generation rate by microsomes exposed to NO decreased significantly as compared to the rate measured in microsomes incubated in the absence of NO. The exposure of the microsomes to the NO donor did not alter the microsomal rate of hydroxyl radical generation. Preincubation of the microsomes with the NO donor affected neither iron reduction rate nor activity of cytochrome c reductase. However, cytochrome P₄₅₀ activity was significantly inhibited after exposure to NO. This inhibition was completely prevented by hemoglobin. The data are consistent with the hypothesis that NO exhibits a potential antioxidant role in the plant cell by decreasing the rate of generation of superoxide anion. Since endogenous NO was detected in homogenates of soybean embryonic axes by EPR studies, this interaction between NO and cytochrome P₄₅₀ in soybean embryonic axes could be a factor of relevance for the control of oxidative stress in vivo.     

Effect of short-chain fatty acids on the ethylene pathway in embryonic axes of Cicer arietinum during germination

The effect of short-chain saturated fatty acids (C₅–C₁₀) on the biosynthesis of ethylene in embryonic axes of chick-pea ( Cicer arietinum L.) seeds was investigated. The emergence of radicle and fresh weight of embryonic axes diminished with increasing number of carbons. The inhibition of germination caused by lower concentrations (1 m M ) of fatty acids (C₅–C₁₀) was partially reversed by exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), whereas exogenous ethylene was able to overcome the inhibitory effect provoked by all concentrations (1–5 m M ) of applied fatty acids (C₅–C₁₀). Ethylene production rates, and enzyme activities of ACC synthase and ACC oxidase decreased concomitantly with the molecular mass and increasing concentration of fatty acids. The inhibitory effect of these acids on ethylene production seems to result not only from a decreased ACC synthesis, but also from an enhancement of 1-malonylamino)cyclopropane-1-carboxylic acid (MACC) synthesis.     

Responses of adenine nucleotides in germinating soybean embryonic axes to exogenously applied adenine and adenosine

The ATP content of soybean (Glycine max [L.] Merr. cv. Kent) axes incubated for 3 hours in 1 mm solutions of adenine and adenosine increased over 100% and 75%, respectively, over axes incubated in water. The increase in ATP was primarily due to the conversion of these purines to nucleotides via the nucleotide salvage pathway. The ATP formed was in a metabolically active pool because label from adenine was incorporated into acid-insoluble material. Adenine also increased the levels of GTP, UTP, and CTP, but not to the extent of the ATP level.     

Prevention of imbibitional injury in low vigor soybean embryonic axes by osmotic control of water uptake

Deterioration of soybean [ Glycine max (L.) Merr. cv. Essex] seeds during accelerated aging at 41°C and 100% relative humidity predisposes the embryonic axis to injury during the initial period of imbibition. This injury was prevented or greatly reduced in severity when excised axes were imbibed on blotters containing 30% polyethylene glycol which slowed the rate of water uptake and when axes were pre-equilibrated to a high moisture level. Rates of water uptake by "high"(no treatment) and "low vigor"(accelerated aged) excised axes were identical. However, high vigor axes tolerated rapid water uptake during early imbition, whereas low vigor axes did not. Leakage of electrolytes during early imbibition was nearly six times greater in low than in high vigor axes. Polyethylene glycol significantly reduced the leakage of electrolytes from both low and high vigor axes. The data are in agreement with the hypothesis that seed deterioration in soybeans involves membrane changes which may predispose embryonic tissues to injury during imbibition. Reduction of the rate of water uptake during the initial period of imbibition would allow extra time for membrane repair or rearrangement, thus permitting the tissues to develop in a more orderly manner. The data indicate that deterioration in soybean seeds involves, at least in part, a decrease in ability of seed axes to tolerate rapid water uptake at the start of imbibition and that this weakness may be compensated by osmotic control of water uptake.