Reactive oxygen species release, vitamin E, fatty acid and phytosterol contents of artificially aged radish (Raphanus sativus L.) seeds during germination

Seeds of Raphanus sativus L. subjected to accelerated ageing were investigated for reactive oxygen species (ROS) release and for content of vitamin E (tocopherol, TOC, and tocotrienol, TOC-3), fatty acids and phytosterols in seed coats, cotyledons and embryonic axes during germination. In unaged seeds, ROS release occurred mainly in seed coats of non-imbibed seeds and in seedlings (48?h of imbibition). TOC and TOC-3 were mainly represented by the ??-isoform, abundant in embryonic axes. Fatty acids were mainly found in cotyledons. In seed coat and embryonic axis, phytosterols consisted mainly of sitosterols. The effects of ageing were mainly visible in embryonic axes at 48?h of imbibition. Deterioration was associated with a decrease in fresh weight increase percentage, germination percentage, ??-TOC and total fatty acid content. An increase in ROS release from seed coats and in ??-TOC, ??-TOC, ??-TOC-3 content in embryonic axis was also observed. The use of ??-TOC and total fatty acids in embryonic axis as parameters of seed quality evaluation during storage was suggested.     

Amino acid uptake and protein synthesis in germinating spores of Saccharomyces cerevisiae.

Spores transferred to germination medium incorporated exogenous lysine into protein within 20 min but required 2-3 to begin incorporation of exogenous proline or alanine. During this time considerable uptake of amino acids into the intracellular pool occurred. Cycloheximide added to the germination medium inhibited incorporation of lysine into protein but did not lessen in accumulation in the pool. Spore germination was inhibited by cycloheximide.     

The Influence of Axis Removal on Protein Metabolism in Cotyledons of Pisum sativum L

The protein metabolism of cotyledons attached to the embryonic axis has been compared with that in cotyledons removed from the axis at the initiation of a 6-day imbibition. Total protein declined in the attached but not in the detached cotyledons. Concurrent with the decline in protein level in the intact cotyledons there was an increased capacity to incorporate exogenously supplied leucine into protein. In contrast, detached cotyledons showed a restricted capacity for protein synthesis. It was demonstrated that ribosomal preparations from cotyledons of intact seedlings contained an increasing proportion of polyribosomes as germination progressed and such ribosomes were active in in vitro amino acid incorporation. Ribosomal preparations from detached cotyledons contained few polyribosomes and had a restricted capacity to incorporate amino acids in vitro. The in vitro incorporation of phenylalanine was stimulated by polyuridylic acid with the stimulation being greatest in ribosomal preparations from detached cotyledons. The results suggest that an axis component may regulate the availability of messenger RNA in the cotyledons during germination.     

Developmental Biochemistry of Cottonseed Embryogenesis and Germination: VIII. Free Amino Acid Pool Composition during Cotyledon Development

The composition of the free amino acid pool in embryonic cotton (Gossypium hirsutum) cotyledons is quite distinct from that of endosperm, and that of germinated, greened cotyledons is quite distinct from that of leaves. During germination (including the precocious germination of immature seeds), the pool expands considerably showing a pronounced accumulation of asparagine. The high level of asparagine found in seedling roots and in the cotyledon vascular exudate indicates that this is the major transported amino acid in germination. There is no pool expansion in the presence of abscisic acid. In the presence of actinomycin D, the pool expands, but an enormous accumulation of glutamine takes place. The composition of the pool at any stage is not related to the composition of the isoacceptor transfer RNA pool, nor to the composition of the storage protein. Anaerobiosis leads to an accumulation of aspartate, alanine, and glycine at the expense of asparagine; however, desiccation does not result in an accumulation of proline. Conspicuously high levels of arginine are maintained through embryogenesis and germination. The levels of individual amino acids are presented as nanomol per cotyledon pair and as per cent of total pool.     

Increased Na+ and Cl− accumulation induced by NaCl salinity inhibits cotyledonary reserve mobilization and alters the source-sink relationship in establishing dwarf cashew seedlings

We studied the NaCl-induced changes in cotyledons and the embryonic axis of establishing dwarf cashew (Anacardium occidentale) seedlings. The salt stress reduced the growth of dwarf cashew seedlings, and this response was related to the inhibition of cotyledonary reserve depletion. Lipid mobilization was inhibited by NaCl due to reduced lipase activity in the emerging and establishing seedlings. Additionally, there was reduced transient starch accumulation in the cotyledons of the salt-stressed seedlings that was associated with lower starch synthase activity at the early developmental stages and inhibited amylolytic and starch phosphorylase activities at the established seedling stage. The NaCl-induced changes in lipid and starch metabolism influenced the soluble sugar content in the cotyledons. Protein mobilization was inhibited by NaCl, and we observed the accumulation of amino acids and the inhibition of proteolytic activity in the cotyledons of the salt-stressed established seedlings. Salinity significantly reduced the free amino acid and reducing sugar contents in the embryonic axes of both emerged and established seedlings, whereas the non-reducing sugar content was affected by this stress only in the established seedlings. The Na⁺ and Cl^? contents progressively increased in the cotyledons and embryonic axis of the seedlings as the salinity increased. We conclude that salt stress inhibits dwarf cashew seedling establishment by inhibiting the mobilization of reserves, an inhibition that was related to increased Na⁺ and Cl^? accumulation in the cotyledons. Additionally, these toxic ions reduced the sink strength of the embryonic axis with regard to the products of cotyledonary reserve mobilization.     

Free Amino Acid, Protein and Water Content Changes Associated with Seed Development in Araucaria angustifolia

The free amino acid, protein, water and dry matter contents were determined during the seed development of Araucaria angustifolia. Soluble and insoluble proteins in the mature seed represent 4.2 % of the fresh matter. The embryonic axis stored the greatest amount of soluble proteins, while cotyledons both with the embryonic axis showed the largest quantities of insoluble proteins in the mature seed. The greatest concentration of free amino acids was detected during the stage when cotyledons start to develop. Glutamic acid, aspartic acid, alanine and serine were predominant in the whole seed while arginine, lysine and γ-aminobutyric acid were present in great amounts only in cotyledons and embryonic axis. Although megagametophyte was important as a source of free amino acids, it was not the major protein storage organ in the mature seed. In the embryogenetic process, the rise of cotyledons is closely related to physiological and biochemical changes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential sensitivity to cadmium in germinating seeds of three cultivars of faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.)

The reserve mobilization was analysed in germinating seeds of faba bean (Vicia faba) exposed to treatment with a toxic cadmium concentration for 4 days. When the behaviours of three cultivars were compared with regard to the germination rate, the following order of sensitivity to cadmium was observed: Aguadulce and Luz de otoño showed 59 and 19% of inhibition from controls, respectively, while no effect was observed in the case of the local Féverole. The growth of embryo radicle was also affected in the same pattern. The differential vulnerability to Cd stress cannot be correlated to shortage in water supply of cotyledons. However, Cd-treated germinating seeds of the most sensitive cultivar (Aguadulce) showed restriction in starch mobilization and decrease in availability of soluble sugars and free amino acids. Moreover, glucose, fructose and amino acids were markedly leaked into the germination medium at the expense of the growing embryonic axis during exposure to Cd. These results provide an indication of the way in which cadmium might impair seed germination.     

Copper Excess Impairs Mobilization of Storage Proteins in Bean Cotyledons

Germination represents a limiting stage of plant life cycle. One of the underlying metabolic activities following imbibition of seed is the reserve mobilization. Seeds of bean (Phaseolus vulgaris L. var. soisson nain hatif) were germinated by soaking in distilled water or 200?μM CuCl(2). Storage proteins breakdown and amino acids freeing from reserve tissues were investigated. Compared to the control, Cu caused a reduction in germination rate, embryo growth, and in mobilization of cotyledonary biomass. The failure in albumin and globulin hydrolysis after the exposure to the pollutant was argued by (1) higher contents of remaining proteins than control ones, (2) persistence of some polypeptide bands resolved by polyacrylamide gel electrophoresis of albumin and globulin-rich fractions, and (3) decrease in the availability of amino acids. Nitrogen starvation in embryonic axis should be associated with the Cu-imposed delay in growth.     

Evidence for the involvement of cytokinins in the regulation of proteolytic activity in cotyledons of germinating beans

The proteolytic activity of an extract of cotyledons of Phaseolusvulgaris increased during the first 7 days of germination, bothwhen casein was the substrate and when the endogenous proteinserved as such. The increase was partially dependent on thepresence of the embryo axis. The effect of the axis was replaceableby kinetin or zeatin, but not by gibberellic acid or IAA. During the period of 3–6 days after the beginning of incubation,the level of amino acids in cotyledons attached to the embryoaxis was lower than in detached ones. No such difference couldbe detected in the first 2 days of incubation. On the otherhand, the influence of the axis on protease activity alreadywas clearly detectable on the second day of germination. Applicationof casein hydrolysate to the seeds brought about an increasein the concentration of amino acids in the cotyledons, but nosimultaneous decrease in the protease level could be detected.We concluded that these results do not confirm the hypothesisthat the influence of the axis on proteolytic activity is dueto its function as a sink for amino acids produced in the cotyledons. (Received August 9, 1979; )     

Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase

 Increased ethylene evolution accompanies seed germination of many species including Pisum sativum L., but only a little is known about the regulation of the ethylene biosynthetic pathway in different seed tissues. Biosynthesis of the direct ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the expression of ACC oxidase (ACO), and ethylene production were investigated in the cotyledons and embryonic axis of germinating pea seeds. An early onset and sequential induction of ACC biosynthesis, accumulation of Ps-ACO1 mRNA and of ACO activity, and ethylene production were localized almost exclusively in the embryonic axis. Maximal levels of ACC, Ps-ACO1 mRNA, ACO enzyme activity and ethylene evolution were found when radicle emergence was just complete. Treatment of germinating seeds with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that endogenous ethylene regulates its own biosynthesis through a positive feedback loop that enhances ACO expression. Accumulation of Ps-ACO1 mRNA and of ACO enzyme activity in the embryonic axis during the late phase of germination required ethylene, whereas Ps-ACS1 mRNA levels and overall ACC contents were not induced by ethylene treatment. Ethylene did not induce ACO in the embryonic axis during the early phase of germination. Ethylene-independent signalling pathways regulate the spatial and temporal pattern of ethylene biosynthesis, whereas the ethylene signalling pathway regulates high-level ACO expression in the embryonic axis, and thereby enhances ethylene evolution during seed germination. Received: 28 September 1999 / Accepted: 27 December 1999     

Developmental changes in endosperm of germinating castor bean independent of embryonic axis

Dry castor bean (Ricinus communis) seeds were cut transversely into halves and the half without the embryonic axis was placed in moist vermiculite at 30 C for 5 days. The development of the endosperm in the half-bean was found to be qualitatively similar to that in the whole seedling in the appearance of various enzymes of gluconeogenesis, the accumulation of glucose and sucrose as the end products of fat utilization, and the development of subcellular structure. It is concluded that during germination of castor bean, the embryonic axis does not directly control the developmental changes in the endosperm.     

Differences in nutrient uptake between the fat body and embryonic primary cultures of silkworm （Bombyx mori）

Nutrition utilization and by-product formation in cultured insect cells has been investigated in several insect cells and has been of great interest to cell culturists and physiologists. In this research the biochemical changes in embryonic and fat body primary cultures of silkworm, Bombyx mori, have been compared. TC-100 medium supplemented with 10% and 20% FBS was used in embryonic and fat body primary cultures, respectively. Medium was renewed every week and the amount of glucose, uric acid, urea, total protein and alkaline phosphatase were measured in the samples from medium of primary cultures using spectrophotometeric methods. All biochemical macromolecules except uric acid showed significant changes. Glucose decreased in embryonic tissues, while in fat body culture its amount increased. Urea accumulation in embryonic culture was higher than in the fat body cultures. Since urea is a by-product, this accumulation could be due to higher utilization of amino acids. Total protein showed considerable changes and was consumed by embryonic culture more than the fat body＇ s. Alkaline phosphatase showed stronger activity in embryonic cells.     

Effects of gibberellic Acid and gold light on germination, enzyme activities, and amino Acid pool size in a dwarf strain of watermelon

Gibberellic acid (GA₃) promotes and continuous gold light inhibits germination of seeds of a dwarf strain (WB-2) of watermelon [Citrullus lanatus (Thunb.) Matsu. and Nakai]. Osmotic inhibition of germination with mannitol in light-grown seeds of WB-2 was only slightly reversed by GA₃ at the concentrations used, whereas, GA₃ substantially relieved osmotic inhibition in dark-grown seeds.

The effects of GA₃ and gold light on development of catalase and invertase activities and on levels of free amino acids in germinating seeds of WB-2 were examined. Light depressed development of catalase and invertase activity. Levels of free amino acids increased more slowly in embryonic axes of light- than dark-incubated seeds, but in cotyledons higher levels of amino acids were maintained in light-grown seeds. GA₃ accelerated the development of catalase activity in whole embryos and invertase activity in embryonic axes, but did not significantly affect invertase activity in cotyledons during germination. GA₃ had little effect on amino acid pools in cotyledons and embryonic axes.

     

Diamine Oxidase Activity During the Germinative and Post-Germinative Growth of the Embryonic Axis in Chickpea Seeds

Diamine oxidase (DAO, EC 1.4.3.6.), which participates in oxidative catabolism of polyamines (PAs), was not detected in the dry viable chickpea (Cicer arietinum L.) seeds. From the time when the embryonic axis acquired an aerobic metabolism, DAO increased concomitantly with the growth of the embryonic axis and at the same time with the deterioration of the cotyledons, although in these organs the values were clearly lower than in the axis. The highest DAO activity in the embryonic axis of seedlings grown for 72 and 96 h was found in the elongation, differentiation and hypocotyl zones, while the lowest was in the apex and plumule. The absence of cotyledons promoted the early appearance of DAO in the embryonic axis. When germination occurred at supraoptimal temperatures (30 – 35 °C), DAO activity was sharply inhibited both in the cotyledons and in the embryonic axis. This inhibition was accentuated further in the presence of cyclohexylamine, an inhibitor of spermidine synthase activity, to such a degree that DAO was undetectable in the cotyledons. DAO inhibition by EGTA and the pronounced reversal induced by Ca²⁺ implies that calcium may be related to DAO activity. The presence of putrescine, spermidine and spermine in the germination medium stimulated DAO activity, although this activity was inhibited when the exogenous PA was cadaverine.     

Oxidative damage and redox change in pea seeds treated with cadmium

Pea seeds (Pisum sativum L.) were germinated by soaking in distilled water or 5mM CdCl2 for 5 days. The relationships among Cd treatment, germination rate, embryonic axis growth, NAD(P)H levels and NAD(P)H oxidase activities in mitochondrial and peroxisomal fractions of cotyledons and embryonic axis were investigated. Heavy metal stress provoked a diminution in germination percent and embryonic axis growth, as compared to the control. A drastic disorder in reducing power was imposed after exposure to cadmium. Heavy metal caused a significant increase in the redox ratio of coenzymes. NADPH oxidase is considered to be oxidative stress-related enzymes. The NAD(P)H oxidase activities were strongly stimulated after Cd exposure. The changes in redox and oxidative properties are discussed in relation to the delay in seed germination and embryonic axis growth.     

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     

Changes in some nitrogenous components during the germination of pea seeds

The changes in major nitrogenous components during the germination of pea seeds have been followed. During the period of rapid axis growth, 3 to 8 days following germination, the nitrogen content of the cotyledons declines rapidly with an accompanying increase of nitrogen in the developing axis. The accumulation of alcohol soluble nitrogen, primarily amino nitrogen, in the cotyledons and axis during germination indicates that the mobilization of nitrogen is facilitated by proteolysis and translocation of the products.     

Effect of an endogenous factor on arginase activity in germinating Bengal gram (Cicer arietinum) seeds

Arginase (EC 3.5.3.1) activity increased in the cotyledon, while it declined rapidly in the embryonic axis of Bengal gram ( Cicer arietinum L. cv. Desi type) seeds during germination. The decrease in enzyme activity in the embryonic axis was accompanied by changes in the properties of arginase in vitro such as decreased stability, increased heat lability and failure to bind to DEAE-cellulose. These alterations were due to the presence of a low-molecular-weight factor in the extract, which was purified and identified as a hexose derivative. During germination the concentration of the factor increased in the embryonic axis, while no detectable level of the factor was present in the cotyledons. We postulate that the factor may have a role in the regulation of arginase activity in vivo.