Influence of the axis on the development of mitochondrial activity in imbibed black gram cotyledons

The respiration rate of mitochondria from detached Vigna mungo L. cotyledons (without the embryonic axis) doubled during the first day after imbibition, whereas that of mitochondria from attached ones increased by only 50%. Contrary to the respiration rate, the respiratory control ratio was higher in attached cotyledons. The activities of enzymes in the mitochondrial fraction from detached cotyledons increased by about 30% during the first day, while those in mitochondria from attached ones changed little. Cycloheximide did not inhibit the development of mitochondrial respiration in either attached or detached cotyledons, although it almost completely inhibited the incorporation of [¹⁴C]-leucine into mitochondrial proteins. Cycloheximide did not retard the increase in the activities of mitochondrial enzymes in detached cotyledons. It is inferred that the development of mitochondria in black gram cotyledons is brought about by repair or activation of mitochondria present in dry seeds and that the axis affects this repair process.     

Interconversion and translocation of free sugars during galactomannan utilization in germinating guar (Cyamopsis tetragonoloba) seed

With the onset of the degradation of galactomannan, the galactose and mannose levels increased in the endosperm. The hydrolysis of galactomannan was more or less complete within the first 3 days of germination. In the cotyledons, sucrose was the predominant free sugar during the period of rapid galactomannan hydrolysis and reducing sugars (glucose + fructose) were present in only 10–20% proportion. The level of soluble acid invertase activity was in the order of embryonic axis > endosperm > cotyledons. On the basis of (a) absence of galactose and mannose, (b) high proportion of sucrose, (c) very fast conversion of [¹⁴C]glucose and [¹⁴C]mannose to [¹⁴C]sucrose and (d) very low levels of both soluble and bound invertases in cotyledons, we conclude that there is an active synthesis of sucrose in this tissue where disaccharide seems to be least hydrolysed during the period of galactomannan mobilization. A rapid hydrolysis of galactomannan in endosperm during early germination resulted in the synthesis of some starch, as a temporary reserve, in cotyledons. When the cotyledons entered the phase of first leaf formation, cotyledonary sucrose was hydrolysed giving rise to invert sugars. In the embryonic axis, the increase in the ratio of reducing sugars to sucrose coupled with a higher level of invertase, compared with sucrose-UDP glucosyl transferase, indicated that free sugars from the cotyledons are translocated to the embryonic axis as sucrose.     

Mobilisation of the major stored reserves in the embryo of fenugreek (Trigonella foenum-graecum L., Leguminosae), and correlated enzyme activities

Changes in total nitrogen, soluble amino nitrogen, lipid and phytate contents, and in the activities of proteinase (pH 7.0), isocitrate lyase and phytase were followed in the endosperm, cotyledons, and axis during germination of fenugreek seeds and subsequent growth of the seedlings. The endosperm is comprised largely of cell-wall galactomannans: the majority of the seed total nitrogen, lipid and phytate (5%, 8%, 0.44% of seed dry weight respectively) is localised within the cotyledons as stored reserves. Germination is completed after 10–14 h from the start of imbibition, but the major reserves are not mobilised during the first 24 h. Then the total nitrogen content of the cotyledons starts to decrease and that of the axis increases; there is a concomitant accumulation of soluble amino nitrogen in both cotyledons and axis. An increase in proteinase activity in the cotyledons correlates well with the depletion of total nitrogen therein. Depletion of lipid and phytate reserves in the different seed tissues constitutes a late event, occurring after 50 h from the start of imbibition, and is coincident with the final disintegration of the endosperm tissue. The depletion of phytate and stored lipids is accompanied by an increase in phytase and isocitrate lyase activity. It appears that the products of lipid hydrolysis are converted by gluconeogenesis to serve as the major source of sugars for the growing axis after the endosperm galactomannan has been completely mobilised.     

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.     

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.     

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.     

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.     

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; )     

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。     

Amide-Linked Indoleacetic Acid Conjugates May Control Levels of Indoleacetic Acid in Germinating Seedlings of Phaseolus vulgaris

We have shown that amide-linked IAA (indole-3-acetic acid) conjugates accumulated to high levels during maturation of bean seeds (K. Bialek and J.D. Cohen [1989] Plant Physiol 91: 775-779). In the present study, we were interested in the fate of these and other IAA conjugates during seed germination. The content of amide-linked conjugates of IAA in cotyledons declined dramatically during the first hours of imbibition. The rate of decline slowed markedly during the period of the resumption of axis growth. The level of amide-linked IAA conjugates in cotyledons remained relatively high after almost 1 week of germination. The decline of IAA conjugates in cotyledons was followed by a steady increase in the content of both free and amide-linked IAA in the embryonic axes. Amide-linked IAA conjugates were also present in the axes cultured on agar after the cotyledons were removed, which suggests that de novo production of these IAA conjugates occurs in the axis of germinating bean seedlings. A comparison of relative amounts of free and conjugated IAA in the axes of intact seedlings and axes cultured on agar showed lower levels of free IAA and higher levels of conjugated IAA in much slower growing isolated axes. These results suggest a more general role for IAA conjugates in the control of seedling growth than simply to serve as a seed storage form of auxin.     

Differences in the activity and distribution of peroxidases from three different portions of germinating Brassica oleracea seeds

Peroxidase (POD, EC 1.11.1.7) activity, cellular localization and isozyme patterns were investigated in the seed integument, cotyledon and embryo axis of Brassica oleracea cv. Cappuccio during pregermination and seedling growth. Seeds started to germinate after 24 h of imbibition. POD activity was localized in the pigmented layer of the integument and in procambial strands of the cotyledon and embryo axis in the first 24 h of imbibition. It was localized in the integumental cells of palisade, pigmented and aleurone layers and in epidermal, meristematic, procambial cells and xylem elements of the root and hypocotyl after 48 h of imbibition. POD activity increased during germination and early seedling growth: in the integument, it reached a maximum value after 72 h of imbibition, in the embryo axis and cotyledons, it increased up to 144 h of imbibition. The increase in peroxidase activity was accompanied by the appearance of new isozymes correlated with the development of seedling tissues. The isozyme profile was characterized by nine peroxidases: isoperoxidase of 50 kDa peculiar to integuments, that of 150 kDa to cotyledons and that of 82 kDa to the embryo axis. During pregerminative phase isozymes of 84 kDa were detected in the integument and cotyledons, of 48.5 kDa in the embryo axis. After germination, peroxidase activity and the complexity of the isozyme pattern increased, suggesting that they play a relevant role after rupture of the integument.     

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)     

Comparison of globulin mobilization and cysteine proteinases in embryonic axes and cotyledons during germination and seedling growth of vetch (Vicia sativa L.)

Vicilin and legumin, the storage globulins of mature dry vetch (Vicia sativa L.) seeds, are found in protein bodies which are present not only in the cotyledons, but also in the radicle, axis and shoot (together, for reasons of simplicity, here called axis). When at 24 h after the start of imbibition (hai) the radicle breaks through the seed coat a major part of the globulins in the axis has already been degraded, whereas in the cotyledons globulin breakdown cannot yet be detected. Globulin mobilization starts with the degradation of vicilin. At 48 hai when globulin mobilization in the cotyledons just begins, the axis is already nearly depleted of globulins. Mobilization of storage globulin is probably brought about by a complex of different cysteine proteinases (CPRs). The papain-like CPR2 and CPR4, and the legumain-like VsPB2, together with their mRNAs, are already present in axes and cotyledons of dry seeds. This means that they must have been formed during seed maturation. Additional papain-like CPRs are formed later during germination and seedling growth. CPR4 and VsPB2 together with their corresponding mRNAs become undetectable as germination and seedling growth proceed. VsPB2 and VsPB2-mRNA are substituted by the homologous legumain-like proteinase B and its mRNA. The composition of stored and newly formed CPRs undergoes developmental changes which differ between axes and cotyledons. It is concluded that storage globulin mobilization in germinating vetch seeds is started by stored CPRs, whereas the mobilization of the bulk of globulin is predominantly mediated by CPRs which are formed de novo.     

Physiological significance of indoleacetic acid and factors determining its level in cotyledons of Lupinus albus during germination and growth

The results demonstrate the profile of the endogenous indole-3-acetic acid (IAA) in the cotyledons of Lupinus albus L. ( L. termis Forssk.) during germination and seedling growth. The auxin level increases markedly after seed hydration, especially during the time of radicle emergence 24 h after the onset of imbibition. This rise is accompanied by a minimal IAA-oxidase activity, formation of indoleacetylaspartic acid (IAAsp) and an increase in the endogenous tryptophan and tryptophan-carboxyl-¹⁴C degradation, though the latter cannot account for the high IAA level detected during early stages of germination. It is believed that cotyledons are a source of IAA to the developing embryonic axis. – The auxin level drops in the cotyledons during seedling growth, 2–18 days after sowing. This is true also for IAAsp and tryptophan-degrading activity of enzyme extracts. Conversely, endogenous tryptophan is increasingly liberated up to day 14, and IAA-oxidase activity climbs to a peak detected on day 12, prior to the appearance of senescence in the cotyledons. – The physiological significance of IAA and the factors regulating its level in the cotyledons during germination and growth are 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.     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

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.     

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     

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.