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.     

Development of Microbodies in Sunflower Cotyledons and Castor Bean Endosperm during Germination

In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2- to 3-fold, and thereafter it remained about constant in light or darkness.     

Subcellular Localization and Developmental Changes of Aspartate-alpha-Ketoglutarate Transaminase Isozymes in the Cotyledons of Cucumber Seedlings

The total activity of aspartate-α-ketoglutarate transaminase in the cotyledons of cucumber (Cucumis sativus L.) seeds increased 17-fold during the first 2 days of germination in darkness and then declined gradually to 20% of the peak activity after 10 days. Exposure of the seedlings to light at day 3 accelerated the decline. The enzyme in the cotyledon extracts from seedlings at various ages was resolved into six distinct isozymes by starch gel electrophoresis. Isozymes 1 and 2 were glyoxysomal isozymes with different developmental patterns. Isozyme 1 followed the developmental pattern of the total enzyme activity in darkness, and was rapidly eliminated upon illumination. Isozyme 2 increased in activity to a peak at day 2 and declined rapidly thereafter, and disappeared completely at day 6; this developmental pattern was independent of light. No major difference in the optimal pH for activity, substrate specificity, and reversibility was observed between isozymes 1 and 2. The combined developmental pattern of isozymes 1 and 2 during germination correlated with that of the glyoxysomes. Isozyme 3 was located in the cytosol and its developmental pattern followed that of the total activity. Isozymes 4,5, and 6 were plastid isozymes and appeared only after 2 days of germination. Unlike many other chloroplast enzymes, the appearance of the chloroplast transaminase isozymes was under temporal control and was independent of illumination. No enzyme activity was detected in isolated mitochondria. The findings illustrate a complicated cellular control system for the appearance of various organelle-specific transaminase isozymes and thus the amino acid metabolism during germination.     

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     

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.     

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.     

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; )     

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)     

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.     

Changes in the levels of enzymes involved in ammonia assimilation during the development of Phaseolus vulgaris seedlings.Effects of exogenous ammonia

Seeds of Phaseolus vulgaris L. cv. White Kidney were germinated and grown either in a nitrogen-free or in an ammonia-supplied medium. The changes in the soluble protein concentration and in the levels of glutamine synthetase (GS, EC 6.3.1.2), NADH–glutamate synthase (NADH-GOGAT, EC 1.4.1.14), ferredoxin-glutamate synthase (Fd-GOGAT, EC 1.4.7.1) and glutamate dehydrogenase (GDH, EC 1.4.1.2), both NADH- and NAD⁺-dependent, were examined in cotyledons and roots during the first 10 days after sowing. Soluble protein declined rapidly in the cotyledons and increased slightly in the roots. GS activity was initially high both in cotyledons and roots but subsequently decreased during seedling growth. Exogenous ammonia hardly affected GS activity. High levels of NADH-GOGAT were present both in cotyledons and roots during the first days of germination. The activity then gradually declined in both organs. In contrast, Fd-GOGAT in cotyledons was initially low and progressively increased with seedling development. In roots, the levels of Fd-GOGAT were higher in young than in old seedlings. Supply of ammonia to the seedlings increased the levels of NADH-GOGAT and Fd-GOGAT both in cotyledons and roots. NADH-GDH (aminating) activity gradually increased during germination. In contrast, the levels of NAD⁺-GDH (deaminating) activity were highest during the first days of germination. Exogenous ammonia did not significantly affect the activities of GDH.     

The Effect of Root Formation on the Levels of Protein, Chlorophyll, RNA, DNA and Carbohydrates in Excised Cotyledons of Cucurbita pepo

In attached cotyledons of Cucurbita pepo L. protein, chlorophyll, RNA, DNA and carbohydrates were found to increase to a maximum level 14 days after commencement of germination, followed by a sharp decline thereafter. Cotyledons excised and planted in a nutrient solution 14 days after sowing showed maximum levels of protein, chlorophyll, RNA, DNA and soluble and insoluble sugars 52 days after planting. The increase in these compounds coincides with the full development of the root system. Excision earlier or later than 14 days resulted in lower levels of protein and other compounds. The decrease in the level after 52 days was parallel to the senescing phase of the cotyledons.     

A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds

Summary Sunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles.     

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.     

Mobilization of Phosphorus in the Cotyledons of Young Seedlings of the Garden Pea (Pisum sativum L.)

Changes in the levels of various phosphorus fractions and ofphytase activity in the cotyledons of young pea seedlings grownin the light have been studied. It is shown that from the onsetof germination there is a lag of several days in the hydrolysisof phytic acid and that this is associated with a low levelof phytase activity in cotyledon extracts. Rapid developmentof phytase during the next few days is accompanied by a rapidincrease in the rate of phytic acid break-down and both reachmaximum levels after 6–7 days from soaking the seed. Theamount of phytic acid in the cotyledons becomes negligible afterabout 15 days and at the same time phytase activity declinesmarkedly. At this point protease activity is at a maximum andthe water content of the cotyledons begins to fall. Removal of the shoot 4 days after soaking the seed caused animmediate decrease in export of phosphorus from the cotyledonsbut did not affect the level of phytic acid for several days.Subsequently there was a small, but significant reduction inthe rate of phytic acid hydrolysis in de-shooted seedlings ascompared with intact plants in spite of the fact that phytaseactivity was not affected for several days. Similar effectswere observed when excised cotyledons were cultured on moistfilter-paper. Control mechanisms for phytic acid hydrolysis are discussedand it is concluded that regulation by the axis of the inorganicphosphate concentration at the sites of phytase activity maybe a means of controlling phytic acid hydrolysis.     

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 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)     

Development of nuclease activity in cotyledons of Pisum sativum L.

Summary The RNA content of pea cotyledons shows little change during the first five days of germination at 22°C. From day five onwards there is a rapid net degradation of RNA, which continues until day thirteen. The DNA content of the cotyledons increases slightly during the first nine days of germination, after which there is a net decrease. Acid and alkaline ribonuclease activities increase markedly between day one and day five, and then decline between day five and day nine. There is a second increase in the activities of both enzymes from day nine onwards. Soluble deoxyribonuclease activity exhibits a single peak, seven days after the onset of germination. The first increase in acid ribonuclease activity is only partially inhibited by cycloheximide at concentrations which severely inhibit protein synthesis.     

Quantitative changes of fatty acids in soybean cotyledons during senescence and regreening

The quantity of total fatty acids in soybean cotyledons during aging, senescence and regreening has been studied. The greatest change in the fatty acid profile during the initial greening of the cotyledons (4–7 days after germination) was a 130% increase in the content of linolenate. Linoleate, as in the case of the other fatty acids, declined in the first 4 days and then increased by 7 days. Following the 10th day after germination, the quantity of palmitate, linoleate, and linolenate decreased continuously through senescence to 20–28% of the maximum quantity of each. When the cotyledons were regreened by removal of the epicotyl 15 or 16 days after germination, linolenate was present in quantities substantially higher than in the senescing cotyledon. On the 22nd day after germination, the quantity of linolenate in regreened tissue was 140% greater than that in senescing tissue of the same age. By contrast, the quantity of linoleate was only 30–40% greater in regreening tissue and the quantity of most of the other fatty acids was similar in both tissues. Similar changes in the quantity of chloroplast fatty acids were observed during this period. Removal of the epicotyl resulted in a higher level of chloroplast linolenate. During aging, the total chlorophyll and the number of chloroplasts reached a maximum on the 10th day and decreased rapidly during senescence. The amount of chlorophyll per chloroplast remained relatively constant during this period whereas the quantity of linolenate per chloroplast decreased during senescence. It is suggested that major structural changes observed in chloroplast membranes may be related to changes in fatty acid composition, but are not dependent on changes in chlorophyll concentration.     

Different Rates of Metabolism of Soybean Proteinase Inhibitors during Germination

During germination, the content of the major Bowman-Birk proteinase inhibitor (BB-E) in the cotyledons of soybean (Glycine max [L.] Merrill cv. Fiskeby V) seeds decreases, becoming a minor form by the sixth day of germination. One of the three other minor species (BB-D) of this inhibitor in the dry seed increases to become the major form in six-day cotyledons. The other two minor species (BB-C and BB-F) also appear to decrease during germination, but at a slower rate compared to the original major inhibitor form BB-E. By 13 days of germination, no distinct Bowman-Birk inhibitor species can be discerned in the cotyledons. The content of the major Kunitz proteinase inhibitor (K-B) also decreases during germination, but much more slowly. One new form of the Kunitz inhibitor (K-A) becomes apparent by the sixth day. By the 13th day, the proportion of the main isoinhibitor to the late-appearing form is approximately two to one. This difference in the rates of disappearance and appearance of isoinhibitor species in the Bowman-Birk and Kunitz proteinase inhibitor classes suggests a differential metabolism of these two types of proteins and a possible difference in function in the soybean plant.     

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.