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The effects of an osmotic shock (300 mM mannitol, -0.67 MPa) or/and increased external hydrostatic pressure on seedlings (42-h-germinated seeds) of radish (Raphanus sativus L. cv. Tondo Rosso Quarantino) were investigated. The osmotic shock did not inhibit H+ extrusion and net K+ uptake, and did not affect the levels of make acid. reducing sugars, sucrose or amino acids or of the energy charge (i.e. the synthesis of energy-rich phosphate bonds). but inhibited the synthesis of proteins. RNA and DNA. measured as incorporation of labelled precursors. When the osmotic shock was applied together with an increased external hydrostatic pressure of the same magnitude (+0.67 MPa). the same metabolic parameters and the inhibition of synthesis of RNA and DNA were not substantially affected. while the inhibition of protein synthesis was practically reversed and the energy charge decreased; the recovery of protein synthesis was not due to a change in labelled leucine uptake capability. Increased external hydrostatic pressure alone decreased the energy charge without affecting the other parameters considered. The possibility that protein synthesis activity is directly controlled by cell turgor pressure (internal hydrostatic pressure) is discussed. [ABSTRACT FROM AUTHOR]

The effect of increasing osmotic values of the medium (mannitol) on the growth and the response mechanisms of seeds of radish (Raphanus sativus L., cv. Ton do Rosso Quarantino) during the early phase of germination was investigated in the presence or absence of fusicoccin (FC). Decreasing the water potential in the medium inhibited the growth and the evolution of protein synthesis and enhanced H+ extrusion, net uptake of K+ and malic acid synthesis. FC, which stimulates these latter functions, counteracted the inhibitory effect of the decreasing water potential of the medium on growth and protein synthesis. Neither in the absence nor in the presence of FC did decreasing water potential of the medium enhance the synthesis of soluble sugars and amino acids to support the osmotic pressure of the seeds. The osmotic and water potentials of the seeds increased during germination. FC made the increase more rapid, while mannitol kept both potentials low. The pressure potentials of the seeds also decreased with time, and both FC and mannitol enhanced this change. If the seeds were without turgor, the development of protein synthesis was blocked. The seeds counteract the effect of decreasing water potentials in the medium by: a) enhancing H+ extrusion (and, as a consequence, wall loosening and transport mechanisms) and the synthesis of malic acid as apparent in the presence of FC; b) regulating the osmotic potentials of the cells (with a lower dilution of the osmotic compounds present in the seeds due to the diminished uptake of water); c) controlling the growth through the effects of a) and b) on the pressure potentials (internal hydrostatic pressure) of the seeds and on protein synthesis. [ABSTRACT FROM AUTHOR]

Entry of the divalent cations Ni2+, Co2+ and Zn2+ into cells of maize (Zea mays L. cv. Dekalb XL 85) root tissue is accompanied by an acidification of the incubation medium, a decrease in both the pH of the cell sap and the level of malate in the cells, and by an inhibition of dark fixation of CO2. K+, on the contrary, induces only a very low acidification of the incubation medium, does not change either the pH of the cell sap or the malate level in the cells, and induces an increase in CO2 dark fixation. Different mechanisms are postulated for the stimulation of proton extrusion by divalent cations and K+. [ABSTRACT FROM AUTHOR]

The changes in several metabolic parameters induced in thermosensitive seeds of Phacelia tanacetifolia Benth. (cv. Bleu Clair) by an inhibitory temperature of 30°C were evaluated and compared to those occurring at the optimum temperature for germination (16°C). The severe inhibition of germination (70%) at 30°C was accompanied by an inhibition of the increase in total RNA and glucose-6-phosphate followed by an inhibition of the increase in DNA, reducing sugars and ATP. Both transcriptional and translational activities occurred at the two temperatures, but with substantial differences. They were higher at 30 than at 16°C after 9 h of inhibition. With time, translational activity became much lower at 30°C, as indicated by the inhibition of the increases in the polysome/monosome ratio and of the levels of activities, evaluated in vitro, of glucose-b-phosphate dehydrogenase, pyruvate kinase, glucokinase and malate dehydrogenase. Transport also occurred at 30°C, as indicated by the positive net flux of K+ into the cells and by the electrochemical equilibrium of mobile K+ between the outside and inside of the cells. There was much less mobile K+ inside the cells at 30 than at 16°C (50 and 13%, respectively, after 24 and 72 h of imbibition). The values of cytoplasmic pH were the same at 16 and 30°C. At 30°C. the vacuolar pH did not change, whereas at 16°C it became mole acidic at 72 h. The possible roles of cytoplasmic K+ concentration and pH in the control of the reactivation of metabolism during germination of Phacelia tanacetifolia seeds are discussed. [ABSTRACT FROM AUTHOR]

To elucidate the inhibitory effect of light on the germination of the negatively photoblastic seeds of Phacelia tanacetifolia Benth. (cv. Bleu Clair), we have compared the changes that occur during the first 24 h of imbibition in the dark and in the light. Here, we give data on transcription products, translation activity, activities of some enzymes involved in the metabolic reactivation of seeds (glucose-6-phosphate dehydrogenase, pyruvate kinase, glucokinase and malate dehydrogenase), and the capability to absorb L-[U-14C]-leucine. During the first stages of imbibition, the changes in the protein pattern and in the mRNA populations that occurred were not related to the light/dark regimes. Differences due to the light treatment did not appear until after 24 h, when new microsomal proteins appeared in the dark and some mRNA populations encoding low molecular mass polypeptides were increased in the light. The translation activity, measured as L-[U-14C]-leucine incorporated into proteins and as the ratio polysomes/monosomes, increased with time in both darkness and light, but to a much higher extent in the dark. The activities of the four enzymes tested were higher in the light than in the dark before 16 h, then decreased in the light and increased sharply in the dark. Together with the behaviour of the increase in the ratio polysomes/monosomes in the dark, this suggests an increased rate of protein degradation in the dark during the period 0-16 h. The absorption of L-[U-14C]-leucine was severely inhibited by light during the whole period studied, as expected since light inhibits the reactivation of the transport mechanisms at the plasma membrane. In seeds imbibed in the dark and then transferred to light, the inhibition by light of absorption and of incorporation into proteins of L-[U-14C]-leucine, as well as the inhibition of the increase in the ratio polysomes/monosomes, was effective before the 22nd h; after which time the control exerted by light disappeared. [ABSTRACT FROM AUTHOR]

31 P nuclear magnetic resonance spectroscopy (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 (&delta) 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 shifted 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 p1 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 24h, 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 reactivation of the transport mechanisms. [ABSTRACT FROM AUTHOR]

The presence of Ni2+ in the incubation medium of maize rout segments (Zea mays L. cv. Dekalb XL 640) induces an early and significant enhancement of the rate of proton extrusion both in the absence and presence of fusicoccin; with time, an inhibition of proton extrusion and a leakage of K+ appear. The inhibition of proton extrusion is accompanied by a decrease in the dark CO2 fixation and by a decrease in the level of malate in the cells, Ni2+ inhibits in vitro phosphoenolpyruvate carboxylase activity of maize roots. The data indicate a correlation between the operativity of the proton pump and that of the malate-pH stat mechanism for the homeostasis of cytoplasmic pH. [ABSTRACT FROM AUTHOR]

A low-molecular-mass protein able to bind GTP in both native and SDS-denaturating conditions was detected in the cytosol of embryos from wheat (Triticum aestivum L.) seeds germinated for 40 h. The protein fulfilled most of the distinguishing criteria common to eukaryotic small GTP-binding proteins. It retained the ability to bind GTP after SDS/PAGE and nitrocellulose blotting. The protein eluted from Sephadex G-200 gel filtration with a Ve/Vo value corresponding to a molecular mass of 18 kDa, whereas on SDS/PAGE the molecular mass was 20 kDa. The native protein, which showed an intrinsic GTPase activity highly sensitive to NaF, bound the guanine nucleotide with high specificity and with a relatively high affinity (Kd ≈ 85 nM). The GTP-binding protein was not detectable in other subcellular fractions; in the microsomal fraction, two other peptides of low molecular mass (23.5 and 21.5 kDa) with GTP-binding activity were detected. These results indicate that in the cytosolic fraction of germinating wheat embryos there is a 20-kDa protein which is biochemically similar to the known small GTP-binding proteins that currently have been detected almost exclusively in the membrane fraction of plant material. [ABSTRACT FROM AUTHOR]

Two forms of EF-1 are present in the high-speed supernatant fraction from wheat embryo homogenate. In embryos from dry seeds EF-1H is 55% of the total amount of EF-1, while alter 40 h of germination this form completely disappears. When germination and protein synthesis are accelerated by means of 6-benzyladenine, the rate of conversion of EF-1H is increased. On the other hand, the block of germination and of the evolution of protein synthesis by abscisic acid, block this conversion; the block of water uptake, that stops germination and causes a decrease m protein synthesis, reverses the conversion of EF-1H to EF-1L, increasing EF-1H from 15% to 40% of the total. [ABSTRACT FROM AUTHOR]

A poly(U)-binding activity is present in the high-speed supernatant fraction of embryo homogenates from wheat seeds. The factor responsible for such activity was found to have a stimulatory effect on the elongation factor 1 (EF-1). It copurifies with EF-1L, the lighter form of EF-1. through Sephadex G-200, DEAE-cellulose, hvdroxvapatiw and poly(U )-Sepharose 4B column chromatography. The two factors could be separated only through a heating step which destroyed EF-l activity whilst leaving most of the poly(U)-binding activity unaltered. [ABSTRACT FROM AUTHOR]