Properties of acid phosphatase from scutella of germinating maize seeds

One acid phosphatase (optimum pH at 5.4) was purified from maize scutellum after 96 hr of germination. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulfate (SDS). The enzyme has a MW of 65 000 ± 4000 as determined by Sephadex G-200 gel filtration and SDS-PAGE. The enzyme contained 16% neutral sugars, and cations are not required for activity. The purified enzyme was not inactivated by DTNB at pH 8. The hydrolysis of glucose-6-phosphate in the presence of 4 mM fluoride and 4 mm EDTA, at pH 6.7 (optimum pH), seems to be catalysed by this acid phosphatase.     

The metabolism of galactose and the raffinose oligosaccharides by germinating bambarra groundnut seeds

Stachyose is present in the highest amount in the soluble sugar fraction of dry bambarra groundnut cotyledons, followed in descending order by raffinose, sucrose and verbascose. During germination in the dark, the stachyose and raffinose content decrease rapidly, but there is little change in the relatively small amount of verbascose present. The sucrose content increases rapidly during the first two weeks and decreases thereafter. Free glucose and fructose were present in the cotyledons after the 7th day and gradually increased in amount with time of germination. Free galactose and other galactose-containing oligosaccharides were not detected in either the dry or germinated bambarra seeds. During germination, galactose was the only identifiable sugar, aside from traces of sucrose, glucose and fructose, in the extracted soluble sugar fraction in the embryonic axes of all ages when the tissue was incubated with D-[1¹⁴C] galactose. With the cotyledons, however, most of the radioactivity was in glucose and fructose during the early period of germination and in sucrose later. A small fraction of the radioactivity was lost as CO₂.     

Kinetic properties of acid phosphatase from scutella of germinating maize seeds

Acid phosphatase purified from maize scutellum showed a kinetic transition from negative cooperativity at pH 5.4, to Michaelian behavior at pH 6.7. The     

Beta-amylase in germinating millet seeds

Beta-amylase (EC 3.2.1.2) was isolated from germinating millet (Panicum miliaceum L.) seeds by a procedure that included ammonium sulfate fractionation, chromatography on DEAE-cellulofine and CM-cellulofine, and preparative isoelectric focusing. The enzyme was homogeneous by SDS-PAGE. The M(r) of the enzyme was estimated to be 58,000 based on its mobility on SDS-PAGE and gel filtration with TSKgel G4000SW(XL), which showed that it is composed of a single unit. The isoelectric point of the enzyme was 4.62. The enzyme hydrolyzed malto-oligosaccharides more readily as their degree of polymerization increased, this being strongest for malto-oligosaccharides larger than 13 glucose residues and very weakly for maltotriose. Amylose, amylopectin and soluble starch were the most suitable substrates for the enzyme. While the enzyme showed some activity against native starch by itself, starch digestion was accelerated 2.5-fold using alpha-amylase, pullulanase and alpha-glucosidase. This enzyme appears to be very important for the germination of millet seeds.     

Polysaccharase metabolism in dormant and germinating Stylosanthes humilis seeds

The levels of certain polysaccharase and glycosidase enzymes were followed during the germination of Townsville stylo seeds. Significant levels of inve     

Induction of polyphenol oxidase in germinating wheat seeds

A 50- and 100-fold increase in the o-diphenolase activity was observed respectively in excised coleoptiles and roots of wheat seedlings after germination for 4–5 days. This increased activity was associated with the appearance of several new multiple forms of o-diphenolase on acrylamide gels. The embryo-less half-seeds dissected from seedlings, however, revealed only a three-fold increase in o-diphenolase activity, without any alteration in the pattern of multiple forms. Cycloheximide substantially inhibited the activity and appearance of multiple forms of o-diphenolase, whereas actinomycin D failed to bring about a similar response. Protein synthesis was probably necessary for the formation of new multiple forms. Unlike o-diphenolase activity which was present in all parts of the seedling, the monophenolase activity was confined to the embryo-less endosperm. A 5–7-fold increase in monophenolase activity was observed in the embryo-less half-seed dissected from the seedling. A single broad band of monophenolase developed on acrylamide gels. This persisted during the early period of seed germination without addition of new multiple forms. No inhibition of monophenolase activity was observed in seeds treated with cycloheximide or actinomycin D.     

Gibberellin estimation and biosynthesis in germinating Hordeum distichon

Gibberellic acid (GA₃) and 13-deoxy-gibberellic acid (GA₇) were identified in extracts of germinating barley as their ¹⁴C-methyl esters. The maximal level of GA₃ was estimated by an isotopic dilution procedure to be 1·5 ng per grain. Germinating barley incorporated 2-¹⁴C-mevalonic acid into several terpenes, whose specific radioactivities were measured, but incorporation into GA₃ could not be detected. Cell-free embryo extracts from germinating barley converted 2-¹⁴C-mevalonic acid into isopentenol, dimethylallyl alcohol, farnesol and squalene, while ¹⁴C-isopentenyl pyrophosphate was incorporated into geraniol, farnesol, geranylgeraniol and squalene. There was no detectable incorporation into the gibberellin intermediate ent-kaurene.     

Purine and pyrimidine bases and nucleosides of germinating Triticum aestivum seeds

Adenine, guanine, uracil and the corresponding nucleosides were identified in mature wheat grain. On germination, contents of these compounds changed in a regular pattern. Amounts of the free bases rose immediately and remained at the increased levels for a short period (3–6 hr). Nucleoside content decreased at the initial phase and increased sharply 12 to 48 hr after germination.     

Protein breakdown and protease properties of germinating maize endosperm

Protein breakdown during germination of maize at 28° is closely correlated with the appearance of protease activity. In the first 2 days of germination, a slight disaggregation of only G₃ glutelins into more simple elements (albumin-globulins) can be observed. Between 2 and 2.5 days, there is extensive breakdown of all protein fractions, the rate of which coincides with the rate of appearance of proteolytic activity. After 2.5 days these phenomena slow down and the bulk of the endosperm proteins disappears. Three acid proteases in endosperm extracts of germinated grain (P₁₁, P₂₁ and P₂₂) have been isolated by affinity chromatography and gel filtration, and partially characterized. P₁₁ (MW 40 000) which is present in the ungerminated grain, cannot hydrolyse prolamins and is insensitive to reducing agents. P₂₁ (MW 36 000) and P₂₂ (MW 12 000), which appear on day 3 of germination, can degrade prolamins in vitro. Reducing agents enhance their activity and prevent their aggregation or denaturation. Comparative assays with different substrates suggest our enzyme preparations are principally endotype proteases with little contaminating carboxypeptidase activity.     

Triacylglycerol and polar lipid metabolism in germinating sea buckthorn seeds

Dark germination of sea buckthorn (Hippophal rhamnoides L.) seeds was characterized by an initial 3-day-long lag-period, when the contents of triacylglycerols (TAGs) and polar lipids (PLs) remained nearly the same due to a retardation in lipid metabolization. Subsequently, TAG content declined rapidly, and by the 10th day of germination, it did not exceed 5% of total lipids. In this case, total saturated (S) and total unsaturated (U) fatty acids (FAs), as well as various TAG types such as S₂U, SU₂, and U₃, were consumed at nearly similar relative rates. At the same time, separate TAG groups, which included one of the individual FAs, such as palmitic (P), stearic (St), oleic (O), linoleic (L), or linolenic (Le), differed from each other in the intensity of degradation. For L- and Le-TAGs, initial and final concentrations were similar, while initial concentrations of St- and O-TAGs by the 10th day of germination increased 2.3- and 1.5-fold, respectively, and as regards P-TAGs, this value decreased 3.5-fold. Thus, P-TAGs considerably exceeded other TAG groups in their consumption rate in seedlings, while St- and O-TAGs ranked below them in this respect. By the 10th day, the absolute level of PLs increased 16-fold due to a de novo formation of lipid membranes of the cells in the course of growth and differentiation of seedling tissues; this increase was accompanied by an increase in the S-FA concentration in PLs and a decrease in the amount of U-FAs.     

Galactomannan utilization in germinating legume seeds

Galactose and mannose, released on hydrolysis of galactomannan in the endosperm of germinating seeds of carob, guar, honey locust and lucerne were absorbed by the cotyledons and further metabolized. In guar, the distribution of ¹⁴C from [U-¹⁴C]-d-glucose, d-mannose and D-galactose into various cotyledon fractions did not provide evidence for preferential channelling of d-galactose into cell wall fractions and d-mannose into glycolysis. Phosphomannoisomerase, which has previously been reported in animals and microorganisms was detected in a number of legume seeds. In honey locust it was located in the cotyledons and its level declined after galactomannan was depleted. This enzyme from lucerne was purified until free of phosphoglucoisomerase and some of its properties are described.     

Starch phosphorylase enzymes in developing and germinating pea seeds

Phosphorylase has been fractionated during development and germination of seeds of smooth and wrinkled-seeded peas. The total phosphorylase levels have been compared. In addition, a number of other pea tissues and other legumes have been examined. Some kinetic properties of the two enzymes present have been measured. Both enzymes have been further purified by affinity chromatography on Sepharose 4B-starch columns and by sequential gel filtration in the absence and presence of amylopectin. The MW and sub-unit structures of the two enzymes have been examined and their possible roles discussed.     

Turnover of starch-bound lysophosphatidylcholine in germinating barley

Free and starch-bound lysophosphatidylcholine (LPC) in germinating barley was isolated and quantified during the first 8 days of germination. During the first 4 days the starch-bound LPC remained at a relatively constant level (ca 0.4,μmol/seed) and then declined during the next 2 days to ca 0.1 μmol/seed. There appeared to be no further loss of this starch-bound lipid on further germination. The decrease in the content of starch-bound LPC is not due to the action of phospholipase C and/or D on the starch lipid because there was no corresponding accumulation of starchbound lysophosphatidic acid or monoacylglycerol. The free LPC remained relatively constant at 0.02 to 0.04 μmol/seed during the entire germination period indicating that the LPC released from the starch during days 5 and 6 is further metabolized. Amylase activity was also measured in the germinating seed and increased 20-fold between days 2 and 4 which just precedes the rapid decline in starch-bound LPC. The starch content of the seed however declined to ca one third of the original value by day 5. LPC represents 65–70 % of the starch-bound lipid phosphorus in the dry seed. Through days 5 and 6 when the loss of LPC is most rapid there is no marked change in this percentage. After 8 days, however, the LPC is only ca 30%. of the starch-bound lipid phosphorus. Ofthe two major populations of starch-bound LPC, the one bearing a linoleyl group appears to decline more rapidly during days 4–6 than does that carrying a palmitoyl group. The role of starch-bound LPC in barley development and germination is discussed.     

Isocitrate lyase in germinating seeds of Prunus dulcis

Isocitrate lyase activity increased in cotyledons of germinating seeds of Prunus dulcis (almond) which had been induced to germinate by either stratification or treatment with gibberellic acid (GA). Germination of embryos, growth of the embryonic axis and activity of isocitrate lyase increased with increasing concentrations of GA from 10^?7 to 10^?3 M.     

ATP-levels of germinating seeds in relation to vigor

Determination of seed vigor was attempted by comparing ATP-levels of deteriorating seed to germination percentage and production of dry matter. Immediately after imbibition of any seed lot investigated, a production of ATP took place. This ATP-accumulation invariably reached a plateau after 6 h of imbibition. Two well germinating seed lots of rape, one of cauliflower and one of sugar beet, were artificially aged by means of elevated storage temperature and humidity. Every second week through 16 weeks of deterioration the levels of ATP, ADP and AMP after 7 h of imbibition were compared with the germination percentage. While ADP- and AMP-contents of germinating seed displayed no change (when imbibed 7 h) during the period of artificial aging, seed deterioration was reflected in the ATP-levels long before loss of viability could be detected by the conventional germination test.
When ATP-levels per seed were related to germination percentage throughout the aging, all four seed lots displayed similar patterns although the absolute figures differed. In contrast to the conventional "per seed' basis, however, ATP per gram seed not only displayed similar deterioration patterns, but the absolute values were also of the same magnitude.     

Gravitational stress on germinating Pinus pinea seeds

In the germination of lipid-rich seeds, the glyoxylate cycle plays a control role in that, bypassing the two decarboxylative steps of the Krebs cycle; it allows the net synthesis of carbohydrates from lipids. The activity of isocitrate lyase, the key enzyme of the glyoxylate cycle, is an indicator of the state of seed germination: stage of germination, growth of embryo, activation and progress of protein synthesis, depletion of lipidic supplies. In order to investigate the effects of gravity on seed germination, we carried out a study on the time pattern of germination of Pinus pinea seeds that were subjected to a hypergravitational stress (1000 g for 64 h at 4 degrees C), either in a dry or in a wet environment, before to be placed in germination plates. During the whole time of germination, we monitored the state of embryo growth and the most representative enzymes of the main metabolic pathways. In treated wet seeds, we observed an average germination of only 20% with a slowdown of the enzyme activities assayed and a noticeable degradation of lipidic reserves with respect to the controls. These differences in germination are not found for dry seeds.     

Glutamine synthetase isozymes in germinating barley seeds

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of ammonia assimilation in higher plants. In the present study the subunit composition and localization of GS in germinating barley ( Hordeum vulgare ) seed have been clarified. Analysis of the GS polypeptide composition by immunoblotting revealed two different polypeptides. A and B, with a molecular mass of 42 and 40 kDa, respectively. In the scutellum subunit A was already present in the ungerminated seed and remained unchanged, whereas subunit B appeared on day 2 and increased about 5-fold during germination. Polypeptide B also appeared later during germination in the aleurone layer, roots and weakly in the etiolated shoots. By immunogold microscopy, GS was detected in the scutellum and the aleurone layer of barley seeds during germination. Subcellular localization of GS on ultrathin cryosections showed that a cytosolic isozyme was present in the scutellum. Our study confirms that only a cytosolic GS is expressed in barley seed, and its subunit composition changes during germination.     

Soluble and particulate lysophospholipase in the aleurone and endosperm of germinating barley

Lysophospholipase was measured in extracts of germinating barley by determining the amount of free [¹⁴C]palmitate released from [1-¹⁴C] 1-palmitoyl-lysophosphatidylcholine (LPC). Soluble and particulate lysophospholipase activity was measured at 1-day intervals in extracts from the aleurone and endosperm of barley seeds germinated for 8 days. The soluble and particulate activities of the aleurone increase approximately in parallel with one another and after 8 days of germination have 20–30 times more activity than at day 1. The activity profiles and the distribution of the activity between the soluble and particulate forms of lysophospholipase in the endosperm are markedly different. With the exception of the first 2 days when the aleurone activity is low, the endosperm activity is less than that associated with the aleurone. The soluble activity increases during the first 3 days and is more active than that of the aleurone. Thereafter it diminishes and remains low. The particulate enzyme, however, increases dramatically between days 4 and 5 and remains moderately high. The fourth and fifth day represent that stage of germination when starch-bound LPC is released in concert with the increase in amylase activity. It is proposed that it is this particulate form of the endosperm activity which may be responsible for maintaining the level of free LPC low in the endosperm of the germinating seed.     

Ribosomes and ribosomal proteins of dry and germinating conifer seeds

The electrophoretic properties of ribosomes and ribosomal proteins of coniferous seeds were determined on polyacrylamide gels. Dry seeds of jack pine (Pinus banksiana Lamb.) contained 80S monoribosomes; polysomes were absent. After 48 hr of imbibition the seeds contained monoribosomes and polysomes. The MWs of the ribosomal proteins of the cytoplasm and chloroplasts were 10 to 82 × 10³ and 9 to 65 × 10³ respectively. Ribosormal proteins from Pinus, Abies, and Pseudotsuga were electrophoretically similar.     

Interaction of light and hormone signals in germinating seeds

Seed germination is regulated by several environmental factors, such as moisture, oxygen, temperature, light, and nutrients. Light is a critical regulator of seed germination in small-seeded plants, including Arabidopsis and lettuce. Phytochromes, a class of photoreceptors, play a major role in perceiving light to induce seed germination. Classical physiological studies have long suggested the involvement of gibberellin (GA) and abscisic acid (ABA) in the phytochrome-mediated germination response. Recent studies have demonstrated that phytochromes modulate endogenous levels of GA and ABA, as well as GA responsiveness. Several key components that link the perception of light and the modulation of hormone levels and responsiveness have been identified. Complex regulatory loops between light, GA and ABA signaling pathways have been uncovered.