Starch degradation in the cotyledons of germinating lentils

Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the α type. Starch is degraded slowly in the first days; during this time, α- and β-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in α-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three α-amylases, and one β-amylase. Based on their heat lability or heat stability, two sets of α-amylase seem to exist in lentil cotyledons.     

STUDIES ON THE SOLUBLE CARBOHYDRATES AND CARBOHYDRATE PRECURSORS IN GERMINATING SOYBEAN SEED

The total soluble carbohydrate fraction of the cotyledons and embryo axis of germinating soybean seedlings declined rapidly during the first 3 days of germination. This depletion began earlier in the embryo axis than in the cotyledon. The total carbohydrate content of the cotyledons of plants grown in light and plants grown in dark was approximately the same for the first 7 days of germination. Between day 9 and 13 the total carbohydrate content of the cotyledons of soybean seedlings grown in dark was higher than that of plants grown in light. The reducing sugar content of light-grown soybean cotyledons increased approximately 5-fold during the first 9 days of germination, whereas that of dark-grown soybean cotyledons increased more slowly during this interval. Reducing sugars in the embryo increased during the early stages of germination until they approximately equalled the total carbohydrate. Between day 4 and 13, oil was depleted more rapidly in the cotyledons of seedlings grown in light than those grown in the dark. The reserve carbohydrates of soybean embryos and cotyledons consisted primarily of low molecular weight oligosaccharides, particularly sucrose, stachyose, and raffinose. These compounds decreased rapidly during germination. The isocitritase activity in the cotyledons of germinating soybean seeds increased rapidly for the first 6 days of germination and then decreased for the next 7 days. The isocitritase activity of plants grown in the dark was higher than that of the plants grown in light at all stages of development, particularly between day 7 and 11.     

Katabolism of plant cytoplasmic ribosomes: RNA breakdown in senescent cotyledons of germinating broad-bean seedlings

Summary Broad-bean cotyledons lost about 80% of their RNA during the first 30 days of seed germination. Both high-molecular-weight ribosomal (r) RNA and low-molecular-weight RNA were degraded at about the same rate. Only small quantities of breakdown products of rRNA were detected within senescent tissue. This finding contrasts strongly with the very rapid cleavage of rRNA and the accumulation of breakdown products when the same cotyledons were homogenised and then incubated at 25°. It would appear, therefore, that there was a barrier between ribonuclease and most ribosomes in vivo which was disrupted by homogenisation. The results are discussed in relation to the mechanism of ribosome katabolism during senescence.     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

The degradation of ribonucleic acid in the cotyledons of Pisum arvense

1. A ribonuclease has been partially purified from the cotyledons of germinating seed of Pisum arvense. 2. The enzyme degrades ribopolynucleotides to adenosine 3'-phosphate, guanosine 3'-phosphate and the cyclic nucleotides cytidine 2',3'-phosphate and uridine 2',3'-phosphate; no resistant ;core' remains. 3. The activity of RNA-degrading enzymes in the cotyledons increases to a maximum during the first 5 days of germination, passes through a minimum around the eighth day, and thereafter increases again. 4. Ion-exchange chromatography of methanol-soluble extracts of cotyledons revealed the presence, amongst other components, of the 2'-, 3'- and 5'-phosphates of cytidine and uridine, the 3'- and 5'-phosphates of adenosine, and guanosine 5'-phosphate. 5. Seed soaked in a solution containing [(32)P]orthophosphate gave a methanol-soluble fraction containing labelled nucleoside 5'-phosphates, but nucleoside 2'- and 3'-phosphates were not labelled. 6. It is believed that the nucleoside 2'- and 3'-phosphates arise by the action of ribonuclease on cotyledon RNA.     

The development of ribonuclease and acid phosphatase during germination of Pisum arvense

1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.     

Far-red mediated polyribosome formation in radish cotyledons

The total ribosome content of radish cotyledons increases during the first 2–3 days of germination both in darkness and under far-red light irradiation; ribonuclease activity is not under phytochrome control during this period. Changes in ribonuclease activity interfere with the analysis of the polyribosomal population. A maximal ratio of polysomes to monosomes is observed 12 h after the onset of far-red light and then it decreases. A 12 h far-red irradiation stimulates the in vivo incorporation of amino acids into proteins. This stimulation persists when seedlings are transferred for 4 h to the dark.     

Ribosomal RNA turnover and the level of ribonuclease activity in the liver of the pregnant rat.

Studies were undertaken on the turnover of ribosomal RNA and on ribonuclease activity in the liver of the pregnant rat in an attempt to explain the accumulation of liver RNA which occurs during the latter half of pregnancy. Between the 15th and 20th day of gestation the rate constant of degradation, biological half-life and daily rate of synthesis of ribosomal RNA were calculated to be 0.0887, 7.81 days and 6.21 mg per liver per 100g body weight respectively. Corresponding values in non-pregnant rats were 0.123, 5.68 days and 3.47 mg per liver per 100g body weight. The increase in RNA was therefore associated with an increase in its rate of synthesis and a decrease in its rate of breakdown. From the 14th day of pregnancy there was a decrease in alkaline ribonuclease activity and a marked increase in the level of alkaline ribonuclease inhibitor. The activity of acid ribonuclease was found to increase and that of acid phosphatase to decrease during this period.     

Development of cytosol and chloroplast aldolases during germination of spinach seeds

The total activity of aldolase (EC 4.1.2.13) and the activities of cytosol and chloroplast aldolase were determined in seeds, cotyledons, primary leaves and secondary leaves of spinach (Spinacia oleracea L., cv. Monopa) during germination. Total aldolase activity in cotyledons increased from low levels to a low maximum in the dark after one week and to a high maximum in white light after three to four weeks and declined thereafter. The activity in primary and secondary leaves started to rise strongly from the 18th and 26th days, respectively, up to the 42nd day of germination. The levels of aldolase activity paralleled the development of leaf area, chlorophyll content and protein content per leaf except that the leaf area of cotyledons continued to increase steadily up to the 42nd day after the maximum of aldolase activity was reached. Resolution of cytosol- and chloroplast-specific isoenzymes by chromatography on diethylaminoethylcellulose indicated that in the light the cytosol enzyme represented approx. 8% of the total activity in cotyledons, primary and secondary leaves throughout germination, and the chloroplast enzyme represented the remaining 92%. Only in cotyledons of dark-grown seedlings was the cytosol aldolase between 25 and 50% of the total activity. Seeds contained almost exclusively a cytosol aldolase. In cotyledons the increase of total activity in the light was specifically the consequence of an increase in chloroplast aldolase while the cytosol aldolase was little affected by light. The light effect was mediated by phytochrome as demonstrated by classical induction and reversion experiments with red and far-red light and by continuous far-red light treatment.Abbreviation DEAE-cellulose diethylaminoethylcellulose     

The Effects of Germination on the Subcellular Distribution of Cholinesterase in Cotyledons of Phaseolus vulgaris

Homogenates of Phaseolus vulgaris cotyledons have been found capable of hydrolyzing acetylthiocholine. The hydrolysis occurs optimally at pH 8.0, and is inhibited by neostigmine but not eserine. Total activity of the enzyme increases about three-fold between the second and third days of germination, and remains high until day 6 before dropping coincident with the appearance of visible morphological symptoms of senescence in the tissue. Fractionation studies have revealed that the enzyme is enriched in preparations of purified cell wall and plasma membrane and is also present in a soluble fraction. The soluble enzyme accounts for more than 70% of the total cholinesterase activity two days after planting but by the fourth day of germination only about 30% of the total activity in the tissue is soluble. During the same period there is a large increase in the specific activities of both the cell wall and plasma membrane enzymes. By the seventh day of germination the particulate and soluble forms of the enzyme both show much reduced activities, but the specific activities of the cell wall and plasma membrane enzymes subsequently increase again. This is thought to reflect breakdown of protein other than cholinesterase in these structures as they in turn become subject to the increasing pressures of senescence. Cholinesterase in plant tissue presumably serves to regulate the endogenous titre of acetylcholine. The behaviour of this enzyme in bean cotyledons has been interpreted in terms of patterns of physiological and ultrastructural change known to characterize this tissue during germination.     

Respiration Rate, Mitochondrial Activity and Mitochondrial Structure in the Cotyledons of Phaseolus vulgaris L. during Germination

Parallel observations have been made on changes in respirationrate, mitochondrial activity, and the fine structure of mitochondria,in the cotyledons of Phaseolus vulgaris germinating at 25^? Cin the dark. While oxygen uptake of the intact cotyledons risesto a peak between the third and fifth days of germination, mitochondrialactivity falls from 36 hours onwards. In most of the storagecells, mitochondrial cristae have become swollen and the matrixhas darkened by the third day. The continulng rise in respirationrate beyond 36 hours could be accounted for by the increasein soluble oxidative activity, which occurs while mitochondrialactivity is falling. Alternatively, it could be mediated bymitochondria from the vascular bundle cells, where swellingand darkening are delayed till after the fifth day of germination,and where there is proliferation of cristae till the fourthday.     

The Development of Glyoxysomes in Peanut Cotyledons and Maize Scutella

The development of glyoxysomes during germination has been studied in isolated peanut (Arachis hypogaea L.) cotyledons and in maize (Zea mays L.) scutella. In peanut cotyledons isocitratase, malate synthetase, and protein associated with the glyoxysomal fraction increase simultaneously from the 3rd to the 8th day of incubation. In scutella of germinating maize seeds the specific activities of isocitratase, malate synthetase, and catalase associated with the glyoxysomes rise until the 4th day of germination and then decline while the total amount of protein present in the fraction stays constant during the first 5 days. If the peanut cotyledons are cultured in 2% glucose, the development of isocitratase and malate synthetase is severely inhibited, but the level of the glyoxysomal protein is not measurably affected.     

Starch degradation during germination of Civer arietinum L. seeds.

The variations in starch and soluble sugar content, in phosphorylase and amylase activities in cotyledons of germinating seeds of Cicer arietinum L. are determined. Results from various experiments prove that the alpha-amylases are chiefly responsible for amylase activity. Phosphorylase plays an important r?le during the first two days of germination, but it is relegated to a secondary position as the amylase activity increases. Disc electrophoresis on polyacrylamide gel shows the existence of a phosphorylase throughout germination, and detects two alpha-amylases after 48 and 96 h germination respectively. The increase in alpha-amylase activity during germination is due to de novo synthesis of the two isoenzymes, since both are inhibited by cycloheximide and actinomyces D. This de novo synthesis depends on some embryo produced factor, unreplaceable either by giberellic acid or by kinetin.     

Distribution and Isoenzymes of Aspartate Aminotrans-f erase in Cotyledons of Germinating Pumpkins

During germination a steady decline in the reserve protein occurred in dark grown pumpkin cotyledons. By 9 days, 80% of this nitrogen reserve was hydrolyzed but only 50 % was removed from the cotyledons. The remaining nitrogen (30 %) was incorporated into water soluble protein which reached a maximum 9 days after germination. The increase in water soluble protein in pumpkin cotyledons parallel the increase in soluble and particulate aspartate aminotransferase (E.C.2.6.1.1.), suggesting that this enzyme is involved in nitrogen metabolism during germination. Little enzyme activity was found in pumpkin tissues other than the cotyledons. Four anodally moving isoenzymes were found in the soluble aspartate aminotrans-ferase fraction and 3 anodally moving isoenzymes were found in the particulate fraction. The slowest moving isoenzymes disappeared first during germination.     

Changes in nitrogen fractions and proteolytic activities in the cotyledons of germinating lentils

Changes in ninhydrin positive material, free amino acids and protein content during germination of seeds of Lens culinaris Med. have been studied. Ninhydrin positive material and free amino acids reached their highest concentration at the fifth day of germination. Total protein which represents 21% of the total dry weight of the lentil cotyledons, suffers a degradation of only 24% in seven days of germination; in the same period of time, reserve proteins underwent a degradation of 69%, legumin being the more abundant at the start, and the more rapidly depleted. Five different classes of proteolytic activities have been reported in lentil cotyledons: caseinolytic, active against the reserve proteins of the lentil cotyledons themselves, aminopeptidase, peptidehydrolase, carboxypeptidase and dipeptidase. The removal of the axis did not seem to exert any significant influence on the enzymatic activity.     

Changes in the Content of Starch and Activities of some Enzymes of Carbohydrate Metabolism in Cotyledons of Germinating Seeds of Voandzeia subterranea

During the germination of the seeds of Voandzeia subterraneaL. Thouars, the content of starch in the cotyledons fell progressively.There was a substantial increase in amylase activity duringthe first 4 d. The activity of starch phosphorylase increasedafter 6 d when there was a rapid decline in amylase activity.The activities of hexokinase, glucose-6-phosphate dehydrogenase,and 6-phosphogluconate dehydrogenase increased, suggesting thepresence of an active phosphogluconate pathway in the cotyledons.     

Association of thymidine and uridine kinase activities with changes in nucleic acid levels during peanut fruit ontogeny

Various stages of pegs, cotyledons and embryonic axes from maturing peanut fruits were examined for their ability to phosphorylate thymidine and uridine. Highest specific activities during peg elongation were found just prior to increases in endosperm nuclei and embryo cell numbers. In the developing cotyledons and axes, the net kinase activities of crude extracts reached a maximum 1–2 weeks before maximal RNA and DNA contents were attained. An exception was the apparent lack of any relationship between uridine kinase activities and RNA levels in developing embryonic axes. The present results support the observation that peanut axes are devoid of thymidine and uridine kinases during the first 24 hr of germination, as fully developed fruits had very low specific activities for both of these phosphate transferases.     

DNA, RNA, protein and heterochromatin changes during embryo development and germination of soybean (Glycine max L.)

DNA, RNA, protein and heterochromatin were measured cytophotometrically in developing soybean (Glycine max) seeds. The average 2C DNA content for the soybean genome was 2.64 pg. The amounts of nuclear DNA in embryo axes showed no significant change during embryo development, whereas the DNA content in cotyledon nuclei increased significantly from 3.58 pg to 5.49 pg. The number of endopolyploid nuclei increased from 26% to 48% and the DNA content from 4.45 to 5.49 pg after cessation of cell division. The changes in RNA and protein content during embryo development were in general similar to those in DNA content. This can be interpreted that increased DNA levels in soybean cotyledons generated during embryogeny increase the protein synthesizing capacity. During the first 15 days of germination, the number of endopolyploid nuclei in cotyledons declined from 46% to 4%, and this decline is interpreted as DNA degradation providing a ready source of nucleosides and phosphates during early embryo growth. A later decline, however, between 15 and 20 days after germination, was age related similar to leaf senescence, because the percentage of endopolyploid nuclei remained unchanged while the number of non-viable cells increased. In senescing cotyledons, 73% and 80% of RNA and protein but only 20% of DNA were lost, as compared to dormant cotyledons. The heterochromatin (condensed chromatin) measurements indicated that nuclei of metabolically inactive dormant and senescent cotyledon nuclei contained an average of 33% more heterochromatin than nuclei from the green cotyledons of seedlings.     

Ureide metabolism during seedling development in French bean (Phaseolus vulgaris)

French bean ( Phaseolus vulgaris ) is a legume that transports most of the atmospheric nitrogen fixed in its nodules to the aerial parts of the plant as ureides. Changes in ureide content and in enzymatic activities involved in their metabolism were identified in the cotyledons and embryonic axes during germination and early seedling development. Accumulation of ureides (ca. 1300 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds. Throughout germination, the total amount of ureides slightly decreased to about 1200 nmol, but increased both in cotyledons and in embryonic axes after radicle emergence. In the axes, the ureides were almost equally distributed in roots, hypocotyls and epicotyls. The pattern of ureide distribution was not affected by the presence of nitrate or sucrose in the media up to 6 days after imbibition. Ureides are synthesized from purines because allopurinol (a xanthine dehydrogenase inhibitor) blocks the increase of ureides. Allantoin and allantoate-degrading activities were detected in French bean dried seeds, whereas no ureidoglycolate-degrading activity was detected. During germination, the levels of the three activities remain unchanged in cotyledons. After radicle emergence, the levels of activities in cotyledons changed. Allantoin-degrading activity increased, allantoate-degrading activity decreased and ureidoglycolate-degrading activity remained undetectable in cotyledons. In developing embryonic axes, the three activities were detected throughout germination and early seedling development. The embryonic axes are able to synthesize ureides, because those compounds accumulated in axes without cotyledons.     

Partial characterization of a protease inhibitor which inhibits the major endopeptidase present in the cotyledons of mung beans

Germination of mung beans (Phaseolus aureus, Roxb.) is accompanied by an increase in the activity of the endopeptidase involved in storage protein metabolism. Enzyme activity in the cotyledons increases 25-fold during the first 5 days of germination. The cotyledons also contain inhibitory activity against the endopeptidase, and this inhibitory activity declines during germination, suggesting that inhibitors may play a role in regulating the activity of the endopeptidase.