Carbamoyl phosphate synthetase activity from the cotyledons of developing and germinating pea seeds

Carbamoyl phosphate synthetase activity was measured in partially purified extracts from cotyledons of developing and germinating seeds of Pisum sativum L. Some properties of the enzyme were established. During cotyledon development, the activity initially increased sharply but decreased during further development. The activity from germinating seeds was only one-tenth of the maximum activity at an early developmental phase. The results are discussed in relation to pea seed development and 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.     

Amylase Activities in Attached and Excised Cotyledons and in Embryonic Axes of Pisum sativum L.

Amylase activity increased in attached cotyledons of peas, Pisumsativum L. var. Bördi, only during imbibition and remainedalmost constant up to 96 h after germination, but in excisedcotyledons the activity increased slightly at first then markedly.In contrast, the content of the reducing sugars was higher inattached cotyledons than in excised ones. A similar inverserelationship has been found between the concentration of reducingsugars in axes (both attached and excised) and amylase activity. The leakage from intact seeds contained more reducing sugarsthan the leakage from excised cotyledons, whereas the amountof proteins released from the cotyledons was four times greaterduring imbibition. This increase in amylase activity in excisedcotyledons is not thought to be the result of axis excision,but to be the result of the leakage of sugars from the cotyledonsduring incubation. These results suggest that the concentration of reducing sugarsmay be a factor that regulates amylase activity in vivo in boththe cotyledons and axis during the germination of pea seeds. (Received August 4, 1982; Accepted December 14, 1982)     

Diverse regulation by sucrose of enzymes involved in storage lipid breakdown in germinating lupin seeds

The regulatory function of sucrose in the activity of lipid-degrading enzymes was investigated in germinating seeds of yellow lupin (Lupinus luteus L.), white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet). The study was conducted on isolated embryo axes, excised cotyledons and seedlings cultured in vitro for 96 h on medium with 60 mM sucrose or without the sugar. The activity of lipase (lipolysis), acyl-CoA oxidase and catalase (fatty acid β-oxidation) was enhanced in all studied organs cultured on medium without sucrose. The activity of cytosolic aconitase (glyoxylate cycle) was stimulated by sucrose in seedling axes and isolated embryo axes, whereas in seedling cotyledons and excised cotyledons, it was inhibited. The regulatory function of sucrose in phosphoenolpyruvate carboxykinase (gluconeogenesis) was observed only in isolated embryo axes and the activity was lower in carbohydrate deficiency conditions. The peculiar features of storage lipid breakdown in germinating lupin seeds and its regulation by sucrose are discussed.     

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.

     

The mobilization of defence mechanisms in the early stages of pea seed germination against Ascochyta pisi

Ascochyta pisi is a necrotrophic pathogenic fungus, which mainly survives between seasons through infected seeds. Defence responses of pea embryo axes to A. pisi were investigated in the heterotrophic phase of seed germination and during the transition from the heterotrophic to the autotrophic phase. Germinated pea seeds, both non-inoculated and inoculated with A. pisi, were cultured in perlite for 96 h. Polarographic studies performed on intact embryo axes of germinating pea seeds infected with A. pisi showed a high respiratory intensity in time from 48 to 96 h after inoculation. Forty-eight-hour embryo axes of germinating pea seeds exhibited the highest respiration rate, which in infected axes was maintained at the following time points after inoculation. Moreover, at 72 and 96 h after inoculation, respiratory intensity was by 64% and 73% higher than in the control. Electron paramagnetic resonance analysis revealed a higher concentration of semiquinone free radicals with g values of g _||?=?2.0031?±?0.0004 and g _⊥?=?2.0048?±?0.0004 in infected axes than in the control. Generation of superoxide anion radical was also higher in infected axes than in the control but stronger at 72 and 96 h after inoculation. Starting from 72 h after infection, the level of Mn²⁺ ions in infected axes decreased in relation to the control. At the same time, the highest activity of superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) was observed in 72-h infected axes. In turn, the activity of peroxidase (EC 1.11.1.7) up to 72 h after infection was lower than in the control. In 48-h infected embryo axes, a very high level of pterocarpan pisatin was observed. Infection of germinating pea seeds with A. pisi restricted mainly the growth of the epicotyl, but did not inhibit the increase in length and fresh weight of root embryo axes versus cultivation time. These results indicate that in pea during the stages of seed germination and early seedling growth, protective mechanisms are induced in embryo axes against A. pisi.     

Asparagine Enhances Starch Accumulation in Developing and Germinating Lupin Seeds

Regulation of starch accumulation in yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupin (L. mutabilis Sweet) developing and germinating seeds was investigated. Research was conducted on cotyledons isolated from developing seeds as well as on organs of germinating seeds, that is, isolated embryo axes, excised cotyledons, and seedling axes and cotyledons. All organs were cultured in vitro for 96 h in different carbon (60 mM sucrose) and nitrogen (35 mM asparagine or 35 mM nitrate) conditions. Ultrastructure observation showed one common pattern of changes in the number and size of starch granules caused by sucrose, asparagine, and nitrate in both developing and germinating seeds. Sucrose increased the number and size of starch granules. Asparagine additionally increased starch accumulation (irrespective of sucrose nutrition) but nitrate had no effect on starch accumulation. Asparagine treatment resulted in a significant decrease in soluble sugar level in all organs of germinating lupin seeds of the three species investigated. The above-mentioned changes were most clearly visible in white lupin organs. In white lupin, starch granules were visible even in cells of sucrose-starved isolated embryo axes where advanced autophagy occurs. The importance of asparagine-increased starch content in the creation of a strong source–sink gradient in developing and germinating lupin seeds is discussed.     

Influence of the Embryonic Axis on Protein Hydrolysis in Cotyledons of Cucurbita maxima

Four-day time course studies of the hydrolysis of cotyledonal storage protein were conducted on intact seeds, seed cotyledons detached from their embryonic axes and on detached cotyledon pairs germinated in the presence of three excised embryonic axes of Cucurbita maxima Duch., cv. Chicago Worted Hubbard. Detached cotyledons germinated alone showed little hydrolysis of the storage protein. However, the amount of protein hydrolysis of the detached cotyledon pairs germinated in the presence of three excised embryonic axes was comparable to the amount hydrolyzed in the cotyledons of intact germinating seeds. Visual growth differences among these treatments were also evident. The size and yellow color intensity of the fourth day treatments were shown to increase in the following order: detached cotyledon pairs alone, intact seedlings, detached cotyledon pairs in the presence of three excised axes. The growth of the hypocotyl and radical was also modified by removal of the cotyledons. These findings suggest that storage protein degradation and cotyledonal growth are controled by the axis. They also indicate that the cotyledons have some influence on the growth of the axes. Time-course studies were made on the hydrolysis of storage protein in the cotyledons of squash and on the distribution of the hydrolytic products during the germination of light- and dark-grown plants. The storage protein was not hydrolyzed during the first 24 hours. It was hydrolyzed at a uniform rate from 1 to 5 days and at a slightly decreased rate from 5 to 7 days. Most of the hydrolytic products were transported to the axial tissue. Proteinase activity in the cotyledons rapidly increased during germination to a maximum level at 2 to 3 days. This was followed by a decline to about the initial value after 7 days.     

Effect of the pea embryo on formation and degeneration of the mitochondrial membrane in cotyledons during germination

The effect of removal of the embryo on the properties of mitochondriain pea cotyledons was investigated. During imbibition of theseeds, mitochondrial activity was enhanced in the cotyledons.In later stages of germination, respiratory activity of themitochondria decreased gradually, and no response of the mitochondriato exogenous ADP was observed. Moreover, considerable activityof cytochrome oxidase wasrecovered in the post-mitochondrialfraction. Mitochondrial fractions isolated from senescent cotyledonscontained only fragmented particles of mitochondria. On theother hand, in cotyledons excised from the seeds and cultivatedunder wet condition, the initial development of mitochondriademonstrated in the attached cotyledons was suppressed. However,respiratory activity of the mitochondria increased in the laterstages of cultivation. The mitochondria remained unfragmentedand responded to exogenous ADP during all stages of cultivation.Also, a change in the density of mitochondria which occurredin the germinating attached cotyledons was delayed in the cultivatedexcised cotyledons. (Received February 27, 1973; )     

Galactosidases in cultivated and wild peas

The level of α- and β-galactosidase was followed in the cotyledons and embryos of germinating seeds of Pisum sativum and P. elatius. α-Galactosidase is preformed in the cotyledons but its activity increases during germination in the embryos. β-Galactosidase activity in embryos increases during germination but shows little change in cotyledons. The possible function of α- and β-galactosidase is discussed.     

Ascorbate and glutathione metabolism in embryo axes and cotyledons of germinating lupine seeds

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.     

A comparative study of water distribution, free radical production and activation of antioxidative metabolism in germinating pea seeds

The aim of this study was to investigate whether there is a relationship between hydration of the embryo axes and cotyledons and the resumption of the oxidative metabolism in both organs of germinating seeds of pea (Pisum sativum L. cv. Piast). Nuclear magnetic resonance (¹H-NMR) spectroscopy and imaging were used to study temporal and spatial water uptake and distribution in pea seeds. The observations revealed that water penetrates into the seed through the hilum, micropyle and embryo axes, and cotyledons hydrate to different extents. Thus, inhomogeneous water distribution may influence the resumption of oxidative metabolism. Electron paramagnetic resonance (EPR) measurements showed that seed germination was accompanied by the generation of free radicals with g₁ and g₂ values of 2.0032 and 2.0052, respectively. The values of spectroscopic splitting coefficients suggest that they are quinone radicals. The highest content of free radicals was observed in embryo axes immediately after emergence of the radicle. Glutathione content decreased during the entire germination period in both embryo axes and cotyledons. A different profile was observed for ascorbate, with significant increases in embryo axes, coinciding with radicle protrusion. Electrophoretic analysis showed that superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) were present in dry seeds and were activated later during germination, especially in embryo axes. The presence of all antioxidative enzymes as well as low molecular antioxidants in dry seeds allowed the antioxidative machinery to be active as soon as the enzymes were reactivated by seed imbibition. The observed changes in free radical levels, antioxidant contents and enzymatic activities in embryo axes and cotyledons appear to be more closely related to metabolic and developmental processes associated with preparation for germination, and do not correspond directly to the hydration of the tissues.     

胚轴对萌发豌豆子叶中淀粉酶活性表达的影响

萌发豌豆的上、下肢轴均能诱导子叶中淀粉酶活性,外源GA和6—BA具有类似胚轴的作用。离体子叶的淀粉酶凝胶电泳只有一条活性极低的酶带,连生子叶中有两条酶带,其中由胚轴诱导新出现了一条活性很高的同工酶带,它的活性受亚胺环己酮的强烈抑制,而受放线菌素D影响不大。推测豌豆子叶中存在淀粉酶的长寿命mRN—A,胚轴和外源激素的作用在于促进mRNA的翻译。     

Control of the formation of amylases and proteases in the cotyledons of germinating peas

Protease activity increased in attached cotyledons of germinated peas (Pisum sativum L. cv. Alaska) as the stored proteins declined but did not increase in excised cotyledons incubated for the same length of time. Cotyledons of seeds germinated in the presence of a casein hydrolysate solution developed less protease activity than did those germinated on water. These results suggest that accumulation of amino acids regulates the protease level in the cotyledons of germinating peas.     

Activation and Inactivation of Alcohol Dehydrogenase in Germinating Pea Cotyledons

Alcohol dehydrogenase (E.C.1.1.1.1.) activity increases markedly in the germinating pea cotyledon in the first 2 days. The activity was not suppressed by the administration of actinomycin D, 6-methylpurine, DL-p-fluorophenylalanine, and D-chloramphenicol. The compounds rather depressed the decrease of alcohol dehydrogenase activity in cotyledons after 3 days of germination. The alcohol dehydrogenase activity in ungerminated pea seeds was activated by treatment with sodium lauryl sulphate, sodium dioctyl sulfosuccinate, dithiothreitol, 2-mercaptoethanol and NADH. The inhibitory effect caused by the extract from 7 day-old cotyledons was diminished markedly in the presence of dithiothreitol and 2-mercaptoethanol, as well as by addition of bovine serum albumin. If dithiothreitol was added to the extraction medium, the enzyme activity from older cotyledons was greatly enhanced.     

Regulation by sucrose of storage compounds breakdown in germinating seeds of yellow lupine (Lupinus luteus L.), white lupine (Lupinus albus L.) and Andean lupine (Lupinus mutabilis Sweet). II. Mobilization of storage lipid

Research of the regulatory function of sucrose in storage lipid breakdown was conducted on isolated embryo axes, excised cotyledons and whole seedlings of three lupine species grown in vitro on medium with 60 mM sucrose or without the sugar. Lack of sucrose in the medium caused significant increase in total lipid content in yellow, white and Andean lupine isolated embryo axes but in Andean lupine seedling cotyledons and excised cotyledons, lipid level was clearly lower in carbohydrates deficient conditions. Sucrose caused no significant effect on fatty acids spectra. The main fatty acid in yellow lupine seeds was linoleic acid, in white lupine oleic acid and in Andean lupine both oleic and linoleic acids. The main phospholipid in organs of three lupine species was phosphatidylcholine. In sugar-deficient conditions, content of phosphatidylcholine and some others phospholipids was decreased. The peculiar features of regulation by sugars of storage lipid breakdown in germinating lupine seeds and induction of autophagy in young carbohydrate starved embryo axes is discussed.     

Fructose 2,6-bisphosphate and the regulation of pyrophosphate-dependent phosphofructokinase activity in germinating pea seeds

The activity of pyrophosphate: d-fructose-6-phosphate-1-phosphotransferase (EC 2.7.1.90, PPi-PFK) in cotyledons and sprouts of germinating pea seeds (Pisum sativum cv Alaska or Green Arrow) increases rapidly during the first 2 to 3 days after imbibition and then declines to a lower activity. The reaction toward fructose 1,6-bisphosphate formation is activated greatly by fructose 2,6-bisphosphate (fru 2,6-P₂); however, the sensitivity of the enzyme's activity to fru 2,6-P₂ activation changes during germination.     

Phospholipids and the Phospholipid Fatty Acids of Germinating Hazel Seeds (Corylus avellana L.)

An analysis of the phospholipids and phospholipid fatty acidsof germinating hazel seeds has been carried out. The phospholipidcontent of the cotyledons and embryonic axes increased duringgermination. Early increases in the relative amounts of phosphatidylglycerol and decreases in the relative amounts of phosphatidylethanolamine occurred in both cotyledons and axes. The laterstages of germination were accompanied by further changes inthe phospholipid composition of the axes. Increases in the degreeof unsaturation of the C₁₈ fatty acids occurred in both tissues.