The influence of the embryonic axis and cytokinins on reserve mobilization in germinating lupin seeds

The influence of the embryonic axis and cytokinins (CKs) onreserve mobilization has been examined in yellow lupin (Lupinusluteus L. cv. JSG 6167) seed during germination and during earlygrowth for up to 9 d in the dark. The study included determinationof starch, soluble sugars, proteins, and amino acid content.Amylolytic and proteolytic enzyme activity was also measuredin untreated cotyledons with intact embryo (attached) or detachedcotyledons (embryo removed), and in detached cotyledons followingtreatment with CKs namely, dihydrozeatin, (diH)Z, and 6-benzylaminopurine,BAP. Generally, the detached cotyledons showed reduced mobilizationand decreased enzymatic activity in comparison to attached cotyledons,indicating the importance of the embryonic axis in this process.However, a rise in protease activity and free amino acid contentwas detected in 9-d-old detached cotyledons suggesting thatthe end products do not necessarily inhibit enzyme activity.While (diH)Z was partially effective in inducing reserve mobilizationand enzymatic activity in detached cotyledons, the effect ofBAP was more pronounced and appeared to replace the embryonicaxis. The embryonic axis of this species has recently been shownto synthesize CKs which are transported to the cotyledons, arehighly stabe and induce cotyledon expansion and chlorophyllsynthesis. The results of the present investigation and previousstudies from this laboratory collectively indicate that theregulation of reserve mobilization in yellow lupin seeds appearsto be mediated, at least in part, by a stimulus, probably aCK, emanating from the embryonic axis. Key words: Lupinus luteus, cytokinins, benzylaminopurine, dihydrozeatin, embryonic axis, lupin seeds, reserve mobilization     

Control of Protein Hydrolysis in the Cotyledons of Germinating Pea (Pisum sativum L.) Seeds

The effects of removal of the shoot or whole axis on the levelsof total, protein, and TCA-soluble nitrogen and on proteaseactivity in cotyledons during germination of garden pea (Pisumsativum L ) seedlings grown in the light have been examined. Removal of the shoot 1 week after soaking the seed caused areduction in the rates of protein hydrolysis and of nitrogentransport from the cotyledons and an increase in the level ofsoluble nitrogen When the entire axis was excised after 4 or9 days there was a great reduction in protein hydrolysis whilethe level of soluble nitrogen remained the same as in de-shootedplants. In the intact plant, proteolytic activity of cotyledon extractsrose to a peak about 15 days after soaking of the seed and thenfell rapidly This fall coincided with a decrease in water contentand in oxygen consumption by the cotyledons. Removal of theshoot or entire axis led to a much smaller and more gradualincrease in protease activity and the subsequent decline inactivity of the enzyme and senescence of the cotyledons werealso delayed. It is concluded that control of protein hydrolysis in pea cotyledonsis not mediated through the level of protease enzymes, as indicatedby the proteolytic activity of tissue extracts, or by the amountof soluble nitrogen compounds accumulated. Protease activityseems to be controlled by the shoot and to be closely linkedto senescence of the cotyledons Protein hydrolysis and transportof nitrogen to the axis, on the other hand, are affected bythe presence of both shoot and root and the axis appears toexert independent control on each of these processes.     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. I. Changes in Endogenous Levels Within the Cotyledons

Ethanolic extracts from the cotyledons of mature dry Phaseolusvulgaris L. seed yielded cytokinin-like activity which co-chromatographedwith zeatin and ribosylzeatin. Under conditions which stimulatedgermination and cotyledon expansion, the level of these cytokininsdecreased rapidly in both intact embryos and excised cotyledons.In the excised cotyledons the decrease was continuous, resultingin very low levels of cytokinin being detected after 4 daysof incubation. With the embryonic axis present, however, theinitial decrease was arrested and reversed after 3 days. Thissuggests that the cotyledons do not synthesize cytokinins butthat these hormones are imported from the embryonic axis, particularlyonce radicle growth is well under way. Phaseolus vulgaris, bean, cotyledons, cytokinins, 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.     

Hormonal control of proteinase activity in squash cotyledons

A crude proteolytic enzyme preparation which hydrolyzes casein was isolated from the cotyledons of squash seedlings. The presence of ethylene diamine tetraacetate or cysteine did not appreciably affect the activity of the preparation. During the course of germination, the level of proteolytic activity increased in the cotyledons of intact embryos through the third day and then decreased. The presence of the embryonic axis during the first 32 hours of germination was a prerequisite for the development of maximum proteolytic activity.     

Influence of Axis Removal on Amino-, Carboxy- and Endopeptidase Activities in Cotyledons of Germinating Vigna mungo Seeds

Endopeptidase (azocaseolytic enzyme) and carboxypeptidase activitiesin cotyledons of germinating Vigna mungo seeds increased until3 days after the onset of imbibition and decreased thereafter.In detached and incubated cotyledons, the endopeptidase activityincreased only a little while the carboxypeptidase activitycontinued increasing even after 3 days of incubation. The activitiesof leucine-aminopeptidase and alanine-aminopeptidase, exceptfor that of one leucine-aminopeptidase isoenzyme relativelyabundantly present in unimbibed dry cotyledons, increased slightlyon the first day and declined during germination. In detachedcotyledons, the activities maintained their initial levels throughoutthe incubation period. When cotyledons were detached from germinatingseedlings on days 2 and 4 then incubated, the endopeptidaseactivity started to decrease just after removal of the axisbut the carboxypeptidase activity increased more markedly thanwhen the axis remained attached. Exogenously supplied GA₃, kinetin,IAA, or their combinations, showed no significant effect onthe developmental patterns of the endopeptidase and carboxypeptidaseactivities in cotyledons. These results are discussed in relationto the role of the axis in controlling peptidase formation incotyledons of germinating V. mungo seeds. (Received November 18, 1983; Accepted February 28, 1984)     

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)     

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.     

Analysis of Lipids of Citrullus lanatus (cv. Sugar baby) During Seed Germination and Seedling Growth

Changes in the dry weight (dry wt.), total and neutral lipids,and fatty acid composition were determined in the cotyledonsand axis of Citrullus lanatus cv. Sugar baby seedlings duringtheir first 12 d growth in the dark, at 25 ? 1 ?C. The major stored reserves were mobilized between days 3 and8. The lipid concentration after day 4 decreased rapidly inthe cotyledons. On the first day the lipid concentration inthe axis was higher than in the cotyledons but decreased rapidlyand from day 5 up to the end of the experiments it remainedat very low values. In the cotyledons, the change in the fattyacid composition was relatively small compared to the significantchange observed in the axis. The neutral lipids of the cotyledonsdecreased sharply between days 2 and 6 while their fatty acidcomposition suffered no significant change. The axis, comparedto the cotyledons, contained a small but concentrated amountof neutral lipids, which decreased up to day 6. Thereafter,an increase was observed. The fatty acid composition of theaxis neutral lipids changed significantly, linoleic and oleicacid decreased while palmitic and linolenic acid increased. Key words: Citrullus lanatus, seed germination, seedling growth, lipids, fatty acids     

Changes in Amino Acid Content and the Metabolism of γ-Aminobutyrate in Cucurbita moschata Seedlings

Ungerminated pumpkin (Cucurbita moschata Poir.) cotyledons contained 30 % of their dry weight as lipid and 26 % as protein, of which 93 % was globulin. There was a rapid degradation of these reserves 4 to 8 days after planting when the cotyledons had their maximum metabolic activity. About half of the mole percent of amino acids found in the globulin reserve was in arginine, glutamate, aspartate, and their amides. The cotyledons had a large soluble pool of arginine, glutamine, glutamate, and leucine. Most amino acids increased steadily in amount in the cotyledons during germination, except glutamine, ornithine, alanine, serine, glycine, and γ-aminobutyrate and these appeared in large amounts in the translocation stream to the axis tissue. Little arginine or proline was translocated. By 10 days, when translocation had decreased, amino acids accumulated. Ornithine, γ-aminobutyrate, and aspartate were rapidly utilized in the hypocotyl, while glutamine, glycine, and alanine accumulated there. Cysteine and methionine levels were low in the reserve, trans-location stream and soluble fractions. γ-Aminobutyrate-U^?14C injected into cotyledons or incubated with hypocotyls was utilized in a similar fashion. The label appeared in citric acid cycle acids and in the amino acids closely related to this cycle, but the bulk of the label appeared in CO₂. The labeling pattern suggests that γ-aminobutyrate was utilized via succinate, and thus entered the citric acid cycle. A close relationship between arginine, ornithine, glutamate, and γ-aminobutyrate exists in the cotyledon with all but arginine being translocated rapidly to the axis tissue where these amino acids are rapidly metabolized.     

Histochemical studies on protease formation in the cotyledons of germinating bean seeds

Summary Protease formation in Phaseolus vulgaris L. cotyledons during seed germination was studied histochemically using a gelatin-film-substrate method. Protease activity can be detected by this method on the 5th day of germination, at approximately the same time that a rapid increase of activity was observed by a test-tube assay with casein as a substrate. At the early stage of germination, protease activity was observed throughout the cotyledon except in two or three cell layers below the cotyledon surface and in several cell layers around the vascular bundles. A highly active cell layer surrounding the protease-inactive cells near the vascular bundles is suggested to be a source of the protease.Brooklyn Botanic Garden Contribution No. 202.     

Changes of aspartate aminotransferase activity, its isoform pattern, and free amino acids content in peanut cotyledons during seed germination

After imbibition of peanut seeds, the concentration of free amino acids in cotyledons increases probably due to hydrolysis of reserve proteins. That increase was accompanied by a stimulation of aspartate aminotransferase (AAT, EC 2.6.1.1) activity especially pronounced between first and sixth day of imbibition. Peanut cotyledons contained several AAT isoforms which could be visualized after polyacrylamide gel electrophoresis (PAGE). Native PAGE of extracts from cotyledons of dry seeds demonstrate three active bands. The intensity of these bands increased reaching a maximum after 6th day of imbibition. An additional band appears transientely on 6th day, but is not visible after 14 days of incubation. A fastest band appears on this latter day. If germination takes place under hypersalinity conditions. the AAT activity severely diminishes and the free amino acid concentration raises. The possibilities of different types of AAT regulation are discussed.     

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.     

Does osmotic regulation of amylase activity in bean cotyledons exist?

The hypothesis that the promotive effect of the embryo axis of the germinating bean seed on amylase activity in the cotyledons is mediated by an osmoregulative mechanism was examined. After 2 days of germination the action of the axis on amylolytic activity was already clearly revealed, whereas at the same time it did not have any influence on osmotic pressure in the cotyledons. When the axis was attached to one cotyledon during 4 days of incubation, osmotic pressure in the cotyledon was lower than its value in the cotyledons of the intact seedling, whereas amylolytic activity was similar in both treatments. It was concluded that the tested hypothesis is not valid in the case of the bean seedling. External osmotic agents brought about a decrease in the level of amylase in the cotyledons, but this does not prove that osmotic changes which are brought about by production of internal metabolites are involved in the regulation of amylase synthesis.     

Gibberellin induces endopeptidase activity in detached cotyledons of Pisum sativum

Endopeptidase activity in cotyledons of 5-day seedlings of Pisum sativum increased rapidly during germination. However, the increase of the activity in detached cotyledons was depressed. We examined whether a growth regulator can be substituted for the embryonic axis on the development of endopeptidase activity. As monitored by an assay with azoalbumin, the development of endopeptidase activity from crude extracts of detached cotyledons appeared to be slightly accelerated by incubation with 10^–5 M GA₃. However, the pattern after gelatin-polyacrylamide gel suggested that the activity induced in detached cotyledons during a 5-d incubation at 10^–7 M GA₃ was the same as that in attached ones during germination for 5 days and an even greater increase in activity was obtained with 10^–5 M GA₃. These results suggest that GA₃ from the embryonic axis induces endopeptidase activity in attached cotyledons at the first stage of germination.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - GA gibberellic acid     

Amino Acids Content in Germinating Seeds and Seedlings from Castanea sativa L

During germination the chestnut (Castanea sativa L.) var ecotype 33 accumulates a large amount of asparagine in the cotyledons. This compound also accumulates in the growing axis:shoots and roots. In the cotyledons, γ-aminobutyrate (GABA) represents a major amino compound during germination and early seedling growth. In young seedlings, 35 days old, arginine predominates over the other soluble amino acids, particularly in roots. Five enzymic activities involved in arginine and GABA have been measured in the storage organ of the seed: arginase and ornithine carbamyltransferase decrease during germination indicating the slowing down of the urea cycle. In contrast, ornithine aminotransferase increases. Glutamate decarboxylase is particularly active about 21 days after imbibition and GABA aminotransferase activity decreases during germination. These two activities are in good agreement with the likely transport of GABA from cotyledons to growing axis. Asparagine, arginine, and GABA are the three amino compounds obviously involved in the mobilization of nitrogen reserves in the germinating chestnut seeds Castanea sativa.     

Regulation of a Sulfur-Controlled Protease in Neurospora crassa

Wild-type Neurospora crassa produces and secretes extracellular protease(s) when grown on a medium containing a protein as its principle sulfur source. Readily available sulfur sources, such as sulfate or methionine, repress the synthesis of the proteolytic activity. Preliminary characterization of the proteolytic enzyme shows it to have a molecular weight of about 31,000, a pH optimum of 6 to 9 with casein as substrate, and esterolytic activity against acetyl-tyrosine ethyl ester with a pH optimum of 8.5. The enzyme activity is completely inhibited by diisopropylfluorophosphate, partially inhibited by ethylenediaminetetraacetate, but unaffected by iodoacetate. The proteolytic activity is temperature labile and is reduced by 75% within 15 min at 60 C. Synthesis of the protease activity is induced by proteins, and to a lesser extent by large-molecular-weight polyamino acids, but not at all by small peptides or amino acid mixtures. During conidial out-growth, the protease(s) first appears at about 8 h and continues to increase while the cells are in an active growth phase. When a low concentration of sulfate is present, the protease(s) is not produced until about 18 h, suggesting that the sulfate must first be used by the cells before the protease is either synthesized or released.     

Hydrolytic enzyme activities in germinating spores ofAdiantum capillus-veneris L.

Changes in hydrolytic enzyme activities were investigated during spore germination ofAdiantum capillus-veneris L. The spores were incubated for 3 days in the dark at 25 C for imbibition, and then germination of the spores was induced by continuous irradiation with red light. At day 2 after onset of the red light irradiation, rhizoids appeared out of spore coats and protonemal cells became visible on the following day. Lipase occurred in dry spores and its activity decreased during 3 days of dark incubation. The activity started to increase when the spore germination was induced by red light irradiation. On the other hand, amylolytic and aminopeptidase activities which were also detected in dry spores decreased continuously during the dark incubation and following the germination process. RNase activity also decreased during 3 days of dark incubation but the activity was retained thereafter at a constant level with or without red light irradiation. Developmental patterns of these hydrolytic enzymes were classified into two groups: One decreased during imbibition and dark incubation but increased after red light irradiation and the other continuously decreased during dark incubation and germination. These results are discussed in relation to compositional changes of cell constitutions such as lipid, sugars, proteins and amino acids during spore germination.     

Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase

The autodigestive proteolytic activity of extracts of cotyledons of mung beans (Phaseolus aureus Roxb.) increased 4- to 5-fold during germination. A similar increase was found in the ability of these extracts to digest added casein or mung bean globulins. The increase occurred after a 2-day lag during the next 2 to 3 days of germination and coincided with the period of rapid storage protein breakdown. To understand which enzyme(s) may be responsible for this increase in proteolytic activity, the hydrolytic activity of cotyledon extracts toward a number of synthetic substrates and proteins was measured. Germination was accompanied by a marked decline in leucine aminopeptidase, while carboxypeptidase increased about 50%. There were no dramatic changes in either α-mannosidase or N-acetyl-β-glucosaminidase, enzymes which may be involved in the metabolism of the carbohydrate moieties of the reserve glycoproteins. The increase in general proteolytic activity was closely paralleled by a 10-fold increase in endopeptidase activity. This activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide. Studies with inhibitors of proteolytic enzymes showed that reagents which blocked sulfhydryl groups also inhibited the rise in general proteolytic activity. Our results suggest that the appearance of a sulfhydryl-type endopeptidase activity is a necessary prerequisite for the rapid metabolism of the reserve proteins which accompanies germination.