Germination and pH of intracellular compartments in seeds of Phacelia tanacetifolia

³¹ P nuclear magnetic resonance spectroscope (NMR) was used to study the response of Phacelia tanacetifolia seeds to dark and light conditions during the first 72 h of incubation. Changes in the chemical shifts (δ) of the pH-dependent ³¹P-NMR signals from the vacuolar and the cytoplasmic orthophosphate pools were correlated with the different incubation conditions. In the dark (favorable to germination), the cytoplasmic pH remained nearly constant over the whole period considered, while the vacuolar pH shitted to more acidic values after the 24th h of incubation. In the light (inhibiting germination), the values of cytoplasmic pH tended to become more acidic than in the dark after the 24th h of incubation, while the vacuolar pH remained practically constant. When seed germination was inhibited in the dark by butyric acid (BA). a permeant weak acid, the values of cytoplasmic and vacuolar pH were similar to those of the ungerminated seeds incubated in the light. When, vice versa, seed germination was promoted in the light by fusicoccin (FC), the values of cytoplasmic and vacuolar pH were similar to those of the dark-germinated seeds. A progressive augmentation of P, metabolism occurred both in the dark and in the light up to the 24th h of incubation. Subsequently, light blocked any further evolution of this parameter. Treatment with butyric acid in the dark again mimicked the effect of light, while FC reversed the negative effect of light. The data show that in Phacelia tanacetifolia seeds germination is linked to a more alkaline cytoplasmic pH. The finding that the light-dependent metabolic inhibition occurs after an early activation of metabolism, i.e. after the first 24 h. suggests that the effects of light on the cytoplasmic and vacuolar pH depend on the early metabolic processes involved in the control of the homeostasis of cell pH and/or on the inhibition of the reactivation of the transport mechanisms.     

Potassium-Dependent Increase in RNA and Protein Synthesis in the Early Phase of Incubation of the Thermodormant Phacelia tanacetifolia Seeds

The seeds of Phacelia tanacetifolia Benth. cv Bleu Clair incubated at 30°C in the dark did not germinate and did not activate K⁺ uptake capacity. The administration of 1 millimolar K⁺ in the early phase of incubation stimulated RNA and protein synthesis. The possible role of K⁺ in promoting the marcromolecular syntheses during the early phase of germination is discussed.     

Effect of butyric acid on the germination of the seeds of Phacelia tanacetifolia

The effect of a weak acid (butyric acid) on the germination of seeds of Phacelia tanacetifolia Benth. (cv. Bleu Clair) has been studied. Butyric acid inhibited the early phase of germination, and the inhibition was correlated to the amount of the per-meant undissociated form present in the incubation medium. The inhibition by butyric acid in the dark was also correlated to a decrease of dark fixation of CO₂ and to a more pronounced decrease in malic acid levels during the early phase of germination; suggesting that the uptake of the uncharged form of this weak acid was followed by a release of H+ into the cytoplasm, leading to a decrease in its pH. The inhibitory effect of butyric acid in the dark on many metabolic events (rise in respiratory activity, levels of reducing sugars, glucose-6–phosphate and malic acid, dark CO₂ fixation, transport activities and macromolecular synthesis) appeared similar to the one of light. Fusicoccin (FC), which directly stimulates the H⁺ pump at the plasmalemma level, ameliorated the effect of butyric acid, as well as that of light, on germination. The bulk of these data is interpreted as suggesting that the mechanism of light inhibition of germination of Phacelia tanacetifolia seeds might be the consequence of a general block of metabolic reactivation due to the presence of an unfavourable (acidic) cytoplasmic pH; which might be explained with the lack of the phytochrome-dependent activation of the H⁺ pump at the plasmalemma level.     

Action of light and gibberellic acid on the growth of excised embryos from Phacelia tanacetifolia seeds

Summary Germination of Phacelia tanacetifolia seeds is inhibited by light. Embryos freed of endosperm grow irrespective of light. However, if they are held in 0.3 M mannitol plus 2% sucrose, light sensitivity is reinstated: growth (i. e., germination) occurs in darkness but not in light. Gibberellic acid (GA₃) releases the inhibition due to light. These results suggest that (a) the photoreceptor site of the seed is in the embryo; (b) GA₃ acts directly on the embryo; and (c) darkimbibed embryos appear to have a higher water-uptake potential.This work was supported in part by the Jane Coffin Childs Memorial Fund for Medical Research, and by the U.S. Atomic Energy Commission under Contract No. AT(11-1)-1338.     

Preformed Messenger RNAs and Early Wheat Embryo Germination

Wheat (Triticum aestivum L.) embryo homogenates have been fractionated into three cell fractions from which RNA was extracted and assayed for mRNA content by in vitro translation and by [³H]polyuridylic acid hybridization. In dry embryos the preformed mRNAs are distributed equally between a rapidly sedimenting “pellet” fraction and a cytoplasmic “ribosomal/subribosomal” fraction. During germination 25 to 40% of the total mRNA becomes polyribosomal. The remaining 60 to 75% is retained in the pellet and ribosomal/subribosomal fractions.     

The Fine Structure of some Dry Seed Tissues Observed after Completely Anhydrous Chemical Fixation

Completely anhydrous fixation with acrolein vapour or osmiumtetroxide vapour was applied to tissues of air-dry seeds: thecoleoptile of wheat (Trilicum aestivum), and plumule and radicleof mung bean (Vigna radiata). Great shrinkage of cells and organelleswas noted, but all the usual organelles could be identified,except for Golgi bodies and (in most cases) ribosomes. The endoplasmicreticulum was very abundant and endoplasmic reticulum tubuleswere closely associated with the storage organelles, namelylipid bodies in the wheat coleoptile, and protein bodies inthe mung bean embryo axis. Aqueous fixation resulted in considerabledistortion of cellular structure. Triticum aestivum L., wheat, Vigna radiata L., mung bean, seed, fine structure, anhydrous fixation     

Rapid Increase in UDP-Glucose during Early Wheat Embryo Germination

The cellular content of UDP-glucose in isolated wheat (Triticum aestivum L.) embryo increases 8-fold during the first 40 minutes of imbibition. An additional 3-fold increase in the amount of UDP-glucose was observed in the next 5 hours of germination. This communication also describes a unique, quantitative method to achieve a high sensitivity in a direct determination of UDP-glucose with Na [³²P]pyrophosphate and UDP-glucose pyrophosphorylase. The sensitivity of the assay for UDP-glucose is 10 picomoles.     

Transcription of Ribosomal and Messenger RNAs in Early Wheat Embryo Germination

Germinating wheat embryos (Triticum aestivum L). synthesize both ribosomal and messenger RNA at the earliest times after the onset of germination. The rates of synthesis of these two RNAs are determined at various stages in germination by an analysis of newly synthesized radioactive RNA on oligo(dT)-cellulose. The rate of messenger RNA synthesis is essentially constant throughout 18 hours of germination, while that of ribosomal RNA synthesis increases steadily, particularly after the onset of cell expansion (6 hours), reaching at 16 to 18 hours, a rate of synthesis between 5- and 20-fold greater than that observed at the earliest stages. The net effect is a relative decrease in the fraction of transcribed high molecular weight RNA that is mRNA. Throughout the first 7 hours of germination, mRNA is 25 to 30% of the transcribed fraction, whereas by 16 to 18 hours it has declined to a level of 4 to 8%.     

The role of darkness, GA and fusicoccin (FC) in breaking photodormancy in Phacelia tanacetifolia seeds

The germination of the negatively photoblastic seeds of Phacelia tanacetifolia Benth. (cv. Bleu Clair) is promoted by gibberellic acid and fusicoccin. In the dark, or in the light in the presence of fusicoccin, seed germination is accompanied by an increase of gibberellic acid-like substances. In these conditions, the inhibition of the synthesis of gibberellic acid-like substances does not prevent seed germination, but it affects the growth and the survival of the seedlings. Seed germination, growth, and survival of seedlings are discussed in relation to phytochrome, fusicoccin, and gibberellic acid-iite substances.     

Embryo and Endosperm Metabolism of Barley Seeds during Early Germination

Embryos detached from germinating barley seeds were immersedin tritiated water or solutions containing ¹⁴C-labelled compounds.Amino acids rapidly became radioactive and later acids of theKrebs cycle. Labelled alanine did not give rise to radioactivesucrose.     

Calcium-calmodulin in germination of Phacelia tanacetifolia seeds: effects of light, temperature, fusicoccin and calcium-calmodulin antagonists

Germination in the dark and at 16°C of photoblastic and thermosensitive seeds of Phacelia tanacetifolia was inhibited when incubated with EGTA and the Ca²⁺-ionophore A 23187; A 23187 in the presence of Ca²⁺ still inhibited germination, but to a lesser extent. Treatments with EGTA or Ca²⁺ at different concentrations in the presence or in the absence of A 23187 did not remove light inhibition. The calmodulin (CaM) inhibitor, calmidazolium, strongly inhibited germination. The specificity of these inhibitors and their effects on seed germination are discussed.
CaM from Phacelia tanacetifolia seeds has been purified and its characteristics (molecular weight, heat and acid stability, kinetics of phosphodiesterase [EC 3.1.4.17] activation) were very similar to those of other plant sources. More than 90% of total CaM was present in the soluble fraction (ca 41 μg g^-1 fresh weight in ungerminated seeds). The CaM level greatly increased in the early phases of seed germination; this increase did not take place when germination was inhibited by light or high temperature. When fusicoccin, a toxin which promotes germination by activating membrane functions, relieved light or high temperature inhibition, CaM increased up to the control value in the dark at 16°C. The parallel increase in CaM and seed germination suggest that CaM plays an important role in the process. Fusicoccin in the dark at 16°C stimulated CaM and fresh weight increase, but not the metabolic reactivation measured as increase in DNA and total RNA levels; at 30°C fusicoccin stimulated the increase in fresh weight and in CaM level, but the increases in DNA and total RNA were very low. These results suggest that the activation of membrane functions with cell enlargement induced by fusicoccin is related to CaM increase.     

The Aril of the Strelitzia reginae Banks Seed: Structure and Histochemistry

Structural and histochemical aspects of the seed aril in Strelitziareginae Banks were studied by bright field and fluorescencemicroscopy, and by SEM. In cross-section, a typical clover leaf-likepattern of the aril threads and of the zone from which thesearise, was demonstrated. This structure is due to cellulosicthickenings of the cell wall. Histochemical tests showed thatthe cell walls appear heavily impregnated with a suberin-likecomponent. Strelitzia reginae seed, aril structure, histochemistry     

The Hilar Region in Leucaena leucocephala Lam. (De Wit) Seed: Structure, Histochemistry and the Role of the Lens in Germination

Structural and histochemical features of the hilar region inthe seed of Leucaena leucocephala Lam. (De Wit) were investigatedby bright-field and fluorescence light microscopy and scanningelectron microscopy. In the dry seeds the lens appeared as anelliptical depression prevailing in size over the hilum andmicropyle. In the imbibed seeds, both naturally softened seedand those requiring softening with boiling water, the lens rosegradually to form a cavity between the palisade layer and themesophyll below. This apoplastic pathway was traced using RutheniumRed dye. In place of the hour-glass cells, the hilar regioncontained abnormal cells, called 'white cells' because theyremained unstained by Toluidine Blue O at pH 4·4. Theapparently opposing characteristics of the lens are discussed.It is hypothesized that the lens acts as a valve. By remainingclosed it hinders the entry of water because of the presenceof a high light-line rich in callose and in lipid-like substancesand to the almost total lack of hydrophilic polysaccharide material.Under favourable conditions for germination, the lens opens,allowing water to enter, through a thin palisade layer and withthe probable intervention of the 'white cells'.Copyright 1995,1999 Academic Press Seed germination, Leucaena leucocephala, hilar region, lens, structure, histochemistry     

K+ uptake in the early phases of germination of the photoblastic and thermosensitive seed of Phacelia tanacetifolia

Germination of Phacelia tanacetifolia Benth. (cv. Bleu Clair) seeds is accompanied in its early phases by the development of the K⁺ uptake capacity. Dormancy due to light and high temperatures and the negative effect on seed germination of a medium with low water potential inhibit the development of potassium uptake. Fusicoccin, which promotes seed germination under all examined conditions, accelerates also the appearance of the K⁺ uptake capacity. The relationship between the development of K⁺ uptake and seed germination in Phacelia tanacetifolia is discussed.     

Glucose Metabolism and Retention of Glucose Metabolites in the Wheat Embryo during Early Germination

There is a lag period in the growth of excised wheat (Triticum vulgare Host.) embryos extending from 0.5 hour to 4.5 hours of their imbibition in water. During this time there is a sharp increase in the embryos' capability to retain several amino and organic acids, to synthesize cell wall components and starch, and to take up glucose. Their capability to metabolize glucose also increases by 30%. Elevation of the ATP content of 1-hour embryos by incubation in 3 mm adenosine is not sufficient to bring about these changes. These changes may be a part of a metabolic adjustment in the embryos which increases their growth potential.     

Rapid Increase in Adenosine 5'-Triphosphate during Early Wheat Embryo Germination

The ATP content of isolated wheat (Triticum aestivum L. var. Polk) embryos increases 5-fold during the first 30 minutes and 10-fold during the first hour of germination to 80% of maximum. The ATP level remains at approximately 800 nanomoles per gram of tissue during the next 15 hours. ADP, AMP, and total adenosine phosphates decrease between 1 and 6.5 hours, while adenylate energy charge increases from 0.6 to 0.8 and remains constant. The rapid increase in ATP during imbibition is consistent with the energy requirement for polyribosome formation and protein synthesis during the first hours of germination. A method for determining nanomole quantities of ATP in tissue extracts by isotopic dilution of γ-³²P-ATP in the hexokinase reaction is outlined.     

Seed and Embryo Germination in Syringa vulgaris and S. reflexa as Affected by Temperature during Seed Development

A nitrogen source was needed for the flowering of Lemna gibba L., a long-day plant, and L. perpusilla Torr., a shortday plant. The level of endogenous amino acids analyzed by an Amino Acid Analyzer, rose during the first few inductive cycles, but was reduced during later stages of the flowering process. Serine and threonine levels increased during the light period and decreased during the dark period in L. perpusilla. Exogenous serine and threonine added to the culture medium at 10^?6M increased the rate of flowering by more than 35% over the controls. Cysteine inhibited flowering, while other amino acids had little or no promotive effect on flowering. Serine and threonine increased flowering rate in L. perpusilla only when added during a dark period of the inductive cycle. The addition of amino acids during a light period not followed by a dark period had no effect on flowering.