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     

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     

Levels of gibberellin-like substances and their possible transport in developing dwarf and normal cultivars of pea (Pisum sativum L.)

The level of gibberellin-like substances was determined in the cotyledons and axis of developing seedlings of dwarf (Little Marvel) and normal (Tall Telephone) cultivars of pea (Pisum sativum L.). The effect of cotyledon removal with GA₃ application on growth was also examined. Greater levels of gibberellin-like substances were observed in the cotyledons of the normal cultivar than the dwarf. This was particularly evident in the cotyledons during the early stages of seedling growth. Subsequently there was a decline in GA levels in the cotyledons. This was coincidental with a rise in GA content in the axis with markedly greater levels in the normal than the dwarf cultivar. Decotyledonated dwarf and normal plants supplied with GA were much taller than the decotyledonated controls. This observation along with those of the gibberellin levels in the cotyledons and axis, provided circumstantial evidence that there may be translocation of gibberellins from the cotyledons to the axis.     

Diamine Oxidase Activity During the Germinative and Post-Germinative Growth of the Embryonic Axis in Chickpea Seeds

Diamine oxidase (DAO, EC 1.4.3.6.), which participates in oxidative catabolism of polyamines (PAs), was not detected in the dry viable chickpea (Cicer arietinum L.) seeds. From the time when the embryonic axis acquired an aerobic metabolism, DAO increased concomitantly with the growth of the embryonic axis and at the same time with the deterioration of the cotyledons, although in these organs the values were clearly lower than in the axis. The highest DAO activity in the embryonic axis of seedlings grown for 72 and 96 h was found in the elongation, differentiation and hypocotyl zones, while the lowest was in the apex and plumule. The absence of cotyledons promoted the early appearance of DAO in the embryonic axis. When germination occurred at supraoptimal temperatures (30 – 35 °C), DAO activity was sharply inhibited both in the cotyledons and in the embryonic axis. This inhibition was accentuated further in the presence of cyclohexylamine, an inhibitor of spermidine synthase activity, to such a degree that DAO was undetectable in the cotyledons. DAO inhibition by EGTA and the pronounced reversal induced by Ca²⁺ implies that calcium may be related to DAO activity. The presence of putrescine, spermidine and spermine in the germination medium stimulated DAO activity, although this activity was inhibited when the exogenous PA was cadaverine.     

No influence of the embryonic axis on the development of diamine oxidase in pea cotyledons

A reexamination has been made for the supposed regulation of pea (Pisum sativum cv Alaska) cotyledonary diamine oxidase (EC 1.4.3.6) activity by the embryonic axis. When dry cotyledons from which the embryo and testa have been removed surgically are imbibed by soaking in water, there is little increase of the enzyme activity during subsequent incubation on filter paper. However, if the dry cotyledons are imbibed and maintained on filter paper from the first, the increase of the enzyme activity is similar to that in the intact seedling. Thus, rapid imbibition of the isolated dry cotyledons is responsible for repression of enzyme development, and a role for the axis need not be invoked.     

Immunocytochemical localisation of lectins in cells of Phaseolus vulgaris L. seeds

Antibodies against pure E₄- and L₄-lectins from the seeds of Phaseolus vulgaris L. raised in rabbits were made monospecific by immunoaffinity chromatography on E₄- or L₄-lectin Sepharose 4B columns. Localisation of lectins in bean seeds was investigated by indirect immunofluorescence and by electron microscopy on sections stained with colloidal gold particles coated with monospecific anti-E₄- and anti-L₄-IgG. In parenchyma cells from the cotyledons both E- and L-type lectins were found inside the protein bodies. Apparently the matrix of all protein bodies contained both types of lectins. On the other hand in vascular and in axis cells the two types of lectins were localised in the cytoplasm, outside the protein bodies. Thus these findings suggest different roles for the lectins: in cotyledons this may be a specific form of N storage, while in vascular and axis cells lectins may have a more direct metabolic part to play.     

Ultrastructural localization of glycinin and β-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method

Summary -Conglycinin (7S globulin) and glycinin (11S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7S globulin could not be detected.     

Influence of the axis on the enzymes of protein and amide metabolism in the cotyledons of mung bean seedlings

The growth of the mung bean (Vigna radiata) seedling is accompanied by the biosynthesis and accumulation of the endopeptidase vicilin peptidohydrolase and the catabolism of the reserve proteins in the cotyledons. If the axis is removed from the dry seeds and the cotyledons incubated on moist sand the accumulation of vicilin peptidohydrolase is reduced by 77% and the catabolism of reserve proteins slowed to 25% of the rate in intact seedlings. The cotyledons and the cotyledon exudate are rich in asparagine and this amino acid accounts for more than half of the reduced nitrogen exported from the cotyledons. Glutamine synthetase and asparagine synthetase, two key enzymes in the pathway of asparagine synthesis, are under temporal control in the cotyledons. Their activities increase 3.5- and 10-fold, respectively, then decline again. These increases in enzyme activity occur to the same extent in excised cotyledons and are prevented when the cotyledons are incubated in 5 micromolar cycloheximide. The results indicate that the axis may control certain key metabolic events in the cotyledons, such as the synthesis of vicilin peptidohydrolase, while many other anabolic activities may not depend on a growing axis.     

Uptake of [2-14C]abscisic acid and distribution of 14C in apple embryos

Pyrus malus L. var. Golden delicious embryos were incubated with (±)-[2-¹⁴C]abscisic acid (ABA) (10^-5 M, 355 kBq mol^-1). After incubations of various durations, the radioactivity was measured in whole embryos, cotyledons, and embryonic axes.With either 48-h or 16-d incubation periods, the uptake of [¹⁴C]ABA depended upon the mode of culture used. The lowest values corresponded to the absorption by the embryonic axis, the highest to the absorption by the distal parts of the two cotyledons. The cotyledons accumulated the main part of the radioactivity under all conditions. Dormant and almost completely after-ripened embryos cultivated for 4 d showed no significant differences in the radioactivity uptake for identical modes of culture. There was a linear relationship between exogenous ABA concentrations (0.5 to 3.10^-5 M) and ABA uptake for embryos cultivated for 4 d with the distal parts of the cotyledons immersed in the medium.Abbreviations ABA abscisic acid. RM, RM⁺, C/2 M, and CM are different modes of embryo cultures: embryonic axis immersed alone (RM), together with the proximal parts of the cotyledons (RM⁺); distal parts of the cotyledons immersed alone (CM); embroyo flat on the medium, the root and the external surface of one cotyledon being in contact with the medium (C/2 M) - PP proximal parts of the cotyledons - DP distal parts of the cotyledons     

Changes in IAA, tryptophan and activity of soluble peroxidase associated with zygotic embryogenesis in Araucaria angustifolia (Brazilian pine)

In A. angustifolia seeds the highest values of freeIAA occurred in the embryonic axis, at the initial phases of development. Thesevalues decreased sharply coincident with the increase of IAA with thedifferentiation of cotyledons and seed elongation. During seed development,tryptophan concentrations varied inversely with free IAA and directly withconjugated IAA. An increase in peroxidase activity was followed by a decreaseinfree IAA in the embryo axis, and in conjugated IAA in the megagametophyte.Megagametophyte tissues did not exhibit significant variation in free IAAduringseed development. Following the stage where cotyledons arise, tryptophandecreased in the megagametophyte and increased in the cotyledons and embryonicaxis.     

Lack of embryonic axis control in Pisum cotyledon mitochondria

Respiratory control ratio (RCR), ADP: O and oxygen uptake by isolated mitochondria from cotyledons of the genus Pisum were studied. It is shown that in P. sativum the embryonic axis has a slight effect on the behaviour of the mitochondria in the cotyledons, accelerating their degeneration. The inducing factor is transferred within 1 hr from the onset of imbibition from the axis to the cotyledon. In P. elatius the embryonic axis completely lacked an effect on the mitochondria in the cotyledons. Mitochondria in P. elatius seemed to be highly organized and not leaky.     

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     

Effect of Salinity on the Activities of RNase, DNase and Protease during Germination and Early Seedling Growth of Mung Bean

Seeds and seedlings of mung bean (Phaseolus aureus Roxb.) were treated separately with NaCl, KCl, Na₂SO₄ and K₂SO₄ solutions of 5 and 10 S/cm conducitivity. The activity of RNase, DNase and protease were estimated in cotyledons, embryo axis, leaves, and roots. Salinity caused inhibition of RNase activity in the cotyledons and roots, but increase in embryo axis and leaves. Activity of DNase was also increased; sodium salt was more effective than potassium irrespective of associated anion. Salinity in general either reduced or had no effect on protease activity in all organs, with the exception of NaCl which doubled it in leaves.     

Enzyme activities in germinating Mungbean (Phaseolus aureus) seeds and their relation with promoter and inhibitors of protein synthesis

Summary Assay of the hydrolyzing enzyme levels, viz.-amylase, adenosine triphosphatase (ATPase), phytase and ribonuclease (RNase) in the germinating Mungbean (Phaseolus aureus) seeds is made to evaluate the degree of substrate breakdown at different periods of germination. In addition to the cotyledons, the principal site of hydrolases, an evidence of their occurrence in the growing embryo has been presented. Enzyme activities in storage tissues, i. e. cotyledons and growing embryo, i. e. axis rise to a maximum sometime between initial and final phase of germination and then declined.Effects of gibberellic acid, chloramphenicol, actinomycin D, and 5-fluorouracil on such enzymes in cotyledons have also been studied to assess how enzyme synthesis is affected by exogenous GA₃ and protein synthesis inhibitors. While GA₃ is able to promote enzyme activities, particularly,-amylase and RNase, protein synthesis inhibitors remarkably inhibited them indicating net enzyme synthesis during germination.     

Phytin is synthesized in the cotyledons of germinated castor-bean seeds in response to exogenously supplied phosphate

Following germination of the castor bean (Ricinus communis L.) seed, levels of phytin decline in both the endosperm and the embryo. However, as seedling growth continues, phytin increase in the latter to a level exceeding that present in the mature dry embryo, while phytin declines concomitantly in the endosperm. It is likely that phosphate mobilized from phytin in the endosperm acts as a substrate for phytin synthesis in the embryo. This is supported by the observation that isolated embryos supplied with phosphate accumulate phytin, particularly in the cotyledons. This increase is enhanced whenmyo-inositol is provided concurrently as a carbon source. Phytin synthesis in the cotyledons of the isolated embryos can occur without the attached axis. Whether initially exposed to exogenous phosphate or not, the isolated cotyledons remain competent in their ability to synthesize phytin for an extended post-germinative period, even though the major reserves are being mobilized at this time.     

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.     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. III. Transport and Metabolism of 8[14C]t-Zeatin Applied to the Radicle

The application of 8[¹⁴C]t-zeatin to the cotyledons of germinatingbean seeds demonstrated that cytokinins are not readily exportedfrom the cotyledons to the embryonic axis during the early stagesof this process. In the cotyledons the applied zeatin is metabolizedextensively to metabolites which are polar and which occur atR_F 0·2–0·5 on paper chromatograms. Thesemetabolites are stable and are not readily exported from thecotyledons. In contrast the metabolites found at R_F 0–0·2are more readily exported. When exported to the radicles andplumules a large proportion of the translocated metaboliteswere converted to compounds which on paper co-chromatographedwith zeatin. This seems to suggest that the embryonic axis hasthe capacity to synthesize cytokinins and that some of the metabolitesformed during its catabolism can also be used for its synthesis. Phaseolus vulgaris, bean, germination, cytokinins, transport, cotyledons