The Changes of Protein Content and Synthetic Activity in Squash Pollen During Germination

The total protein content of squash (Cucurbita moschata Duch.) pollen decreased gradually during in vitro germination. It was caused by the release of wall proteins and part of the cytoplasmic proteins. The release of the pollen wall proteins was not dependent on germination, it was a passive diffusion process. However, the cytoplasmic proteins did not release until the pollen germinated, a fraction of them was synthesized de novo during germination. The RNA and protein synthetic activities initiated soon after in vitro pollen germination. The RNA synthesis decreased during germination. As about half the activity was inhibited by α-amanitin, mRNA might be the major RNA synthesized de novo. The total protein synthesis increased during germination, almost all of this synthesis was inhibited by cycloheximide, and partially by α-amanitin, but it was not affected significantly by actinomycin D. These results indicated that both stored and de novo synthesized mRNA might play a role in the protein synthesis. The content of stored mRNA of squash pollen was about 11-3 pg/grain as measured by UV absorption after its purification from total RNA (2440 pg/grain) by oligo (dT)-cellulose affinity chromatagraphy. Both cycloheximide and α-amanitin inhibited pollen tube growth in vitro. Actinomycin D and tunicamycin inhibited pollen germination in the first hour, however, no reduction ,of the tube length was observed later. Cyclohex,nide inhibited the pollen germination and tube elongation in vivo, that fitted well with the in vitro results. According to these results, it was suggested that the de novo syntheses of mRNA and protein were neccessary for the maintenance of pollen tube growth.     

Studies on the regulation of the branched chain amino acyl-tRNA synthetases of the fungusNeurospora crassa

Summary The specific activities of the branched chain amino acyl-tRNA synthetases from the cytosolic and mitochondrial fractions ofN. crassa were low in dormant conidia and increased during germination, reaching a maximum 8 h after inoculation. This stage of development is characterised by high rates of many other cellular activities.The increases in activity of synthetases of both cytosol and mitochondria are inhibited by cycloheximide indicating that they are synthesized on cytoplasmic ribosomes. The mitochondrial synthetases show a stimulation of their specific activity when mitochondrial RNA and protein synthesis are inhibited by either ethidium bromide or chloramphenicol suggesting that a mitochondrial translation product regulates the synthesis of the mitochondrial synthetases.The activities of amino acyl-tRNA synthetases are dependent on energy production. When respiration is uncoupled from oxidative phosphorylation, synthetase specific activities decrease although the activities of other mitochondrial enzymes like NADH-dehydrogenase increase. This phenomenon suggests that more than one mechanism regulates the synthesis of mitochondrial proteins which are formed on cytoplasmic ribosomes.The synthesis of branched chain amino acyl-tRNA synthetases ofNeurospora is neither repressed by their cognate amino acids, nor is there inhibition by the precursors of these amino acids, as has been observed in other amino acyl-tRNA synthetases of various organism includingNeurospora.     

The effect of ethylene on RNA and protein synthesis in germinating Manketti (Ricinodendron rautanenii Schinz) seeds

Amino acid uptake and protein synthesis were monitored in the embryonic axis, cotyledons and endosperm of manketti seeds (Ricinodendron rautanenii Schinz) during imbition, dormancy and dormancy-breaking by ethylene. Protein synthesis increased in all tissues within 48 h of imbibition and was strongly inhibited by cycloheximide and actinomycin D. No marked increase in protein synthesis was observed following ethylene treatment suggesting that qualitative rather than quantitative changes in protein synthesis were associated with dormancy-breaking. Seed germination was relatively insensitive to treatment with actinomycin D in marked contrast to the inhibition observed with cycloheximide.It is suggested that dormancy-breaking by ethylene may require protein synthesis but not DNA-dependent RNA synthesis.     

Regulation of De-N-Glycosylation Enzymes in Germinating Radish Seeds

The activities of the de-N-glycosylation enzymes endo-N-acetyl- [beta]-D-glucosaminidase (ENGase; EC 3.2.1.96) and peptide-N4- (N-acetyl-[beta]-D-glucosaminyl) asparagine amidase (PNGase; EC 3.5.1.52) were monitored during germination and postgerminative development in radish (Raphanus sativus L. cv Flamboyant). The ENGase activity was detected only during postgermination, whereas the PNGase activity was present at high levels in both stages. When germination was inhibited with abscisic acid or cycloheximide, PNGase activity was detected at a basic level and ENGase activity was not detected at all. PNGase is present as an active protein in dry seeds and is apparently synthesized during seed formation. Conversely, the absence of ENGase in dry seeds suggests that its activity is dependent on the protein synthesis that occurs during and after germination. Treatment with gibberellic acid confirmed the production of both de-N-glycosylation enzymes after germination, and demonstrated a temporal delay between the production of the two enzymes during this period. Our results suggest that the two de-N-glycosylation enzymes are differentially regulated during plant development.     

Synthesis of the peptide-transport carrier of barley scutellum during the early stages of germination

Development of peptide-transport activity in the scutella of isolated barley (Hordeum vulgare l. cv. Maris Otter, Winter) embryos is shown to increase rapidly after about 15 h of imbibition, with the bulk of the transport activity being expressed by 24 h. This development is prevented by treatment of 15 h embryos with cycloheximide. Protein synthesis is found to increase in a closely related manner and also to be abolished by cycloheximide. Measurement of the incorporation of bound [³⁵S]methionine by 15 to 21-h embryos indicates that de-novo protein synthesis during this period is greater in the scutellum than in the embryonic axis. Previous studies have shown that the peptide-transport system possesses essential dithiol groups, probably located at the substrate-binding site (Walker-Smith and Payne 1983 b, 1984b). Treatment of 15-h embryos with the non-penetrant thiol reagent p-chloromercuribenzene sulphonic acid did not affect development of peptide-transport activity during the following 6 h, whereas with 3-d embryos identical treatment inhibited uptake almost completely during a subsequent 6-h period. Radioautography revealed that amongst the proteins synthesised during this early phase of germination and labelled in vitro with [³⁵S]methionine some are found within the epithelial plasmalemmae of the scutellum, which is the location of the peptide-transport carrier identified previously by externally labelling with a radioactive thiol reagent. The results provide evidence that protein(s) of the peptide-transport system are synthesised and inserted into the scutellum during early germination, allowing the system to play a major role in the nitrogen nutrition of the embryo.Abbreviations Gly Glycine - Phe phenylalanine     

Effect of Purine and Pyrimidine Analogues on Growth and RNA Metabolism in the Soybean Hypocotyl-the Selective Action of 5-Fluorouracil

The effects of several base analogues and cycloheximide on RNA synthesis, protein synthesis, and cell elongation were studied in excised soybean hypocotyl. None of the pyrimidine analogues tested affected growth or protein synthesis; only 5-fluorouracil appreciably inhibited RNA synthesis. 8-Azaguanine and 6-methylpurine markedly inhibited RNA and protein synthesis and cell elongation. Cycloheximide effectively inhibited both cell elongation and protein synthesis.The results show that 5-fluorouracil selectively inhibited ribosomal and soluble RNA synthesis without affecting the synthesis of D-RNA. These results indicate that the requirement for RNA synthesis to support continued protein synthesis and cell elongation is restricted to the synthesis of D-RNA.5-Fluorouracil was incorporated into all classes of RNA in a form believed to be 5-fluorouridylic acid.Cycloheximide markedly inhibited the accumulation of ribosomal RNA, but the results indicate that CH did not inhibit, per se, the synthesis of ribosomal RNA. The accumulation of newly synthesized D-RNA was only slightly affected by cycloheximide. These results show that the inhibition of cell elongation by cycloheximide correlates with the inhibition of protein synthesis, but not with the effect on RNA metabolism.     

Nitrate assimilation during the anaerobic germination of rice: expression of ferredoxin-dependent glutamate synthase

Ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) is the last enzyme involved in the pathway of nitrate assimilation in higher plants. This paper describes the synthesis and expression of the enzyme in anaerobic coleoptiles of rice (Oryza sativa L.) and its regulation by exogenous nitrate. The activity of Fd-GOGAT was strongly inhibited by cycloheximide between 4 and 9 d of anaerobic germination. The addition of nitrate slightly increased, in the first 5 h, the specific activity of Fd-GOGAT as well as the amount of a 160-kDa protein specifically immunoprecipitated with anti-Fd-GOGAT serum. Northern blot analysis, performed with a specific riboprobe, showed the presence of mRNA of the expected size and the inductive effect of nitrate. The role of Fd-GOGAT is discussed in relation to the anaerobic assimilation of nitrate by rice coleoptiles.Abbreviations CHX cycloheximide - Fd ferredoxin - GOGAT glutamate synthase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase The authors wish to thank Dr. J. Turner (Rothamsted Experimental Station, Harpenden, UK) for providing Fd-GOGAT antibody and Dr. H. Sakakibara (Nagoya University, Nagoya, Japan) for Fd-GOGAT clone. This research was supported by the National Research Council of Italy, special project RAISA, sub-projekt N. 2, paper N. 2174.     

Cell wall carbohydrates in Phycomyces blakesleeanus burgeff

The carbohydrate composition of the cell walls from spores, mycelium and sporangiophores of Phycomyces blakesleeanus was analyzed. Spore wall polysaccharides contained over 50% glucose, about 20% uronic acids, 10% mannose and 10% amino-sugars. During the growth of the hyphae amino-sugars became the main carbohydrate (45%); uronic acids contributed some 25%, glucose and fucose 10% and galactose nearly 6%. Sporangiophores contained almost 90% aminosugars and some 6% uronic acids. Traces of rhamnose were found in all wall preparations. A similar picture emerged from studies on the incorporation of [U-¹⁴C]-glucose into wall materials.Furthermore we looked for a GDP-fucose synthesizing system and found an increasing activity during early germination. This rise in activity was inhibited by cycloheximide but not by 5-fluorouracil.     

Sull'evoluzione Dei Mitocondri Nell'endosperma di Semi di Ricino in Germinazione

Abstract

On the behavior of mitochondria in the castor bean seed endosperm during the early phases of germination. — In the endosperm of the castor bean seed the oxidative activity and the protein nitrogen contents of the mitochondrial fraction markedly increase during the first period of germination (Beevers and coworkers). The activation of the mitochondrial system is paralleled by a similar increase of the activity of several soluble enzymes; the latter process is severely depressed by protein synthesis inhibitors (Cornaggia, Aberghina).

The present research is aimed to understand at what extent phenomena of activation and/or, respectively, of « ex novo » synthesis are responsible of the increase of mitochondrial activity. The following aspects of the mitochondrial behavior during the early period of germination were investigated:

a) Changes in the activity of cytochrome oxydase, malate dehydrogenase and of the succinate-citochrome reductase system.

b) Changes in the morphology of mitochondria and other particulated cell structures, as revealed by electron microscopy.

In the mitochondrial preparation all of the three enzymatic activities investigated were found to increase rapidly during the first days of germination. The increase during the first 24 hours was almost as large when measured as specific activity (activity per mg protein in the mitochondrial fraction) than when measured on an absolute (i.e. per seed) basis; moreover, it was not significantly inhibited by puromycin or by actinomycin. The increase of the three activities during the following period of germination (second-third day) was accompanied by an increase of the protein nitrogen (per seed) in the mitochondrial fraction, and was consistently depressed by the protein synthesis inhibitors.

In the mitochondrial preparation all of the three enzymatic activities investigated were found to increase rapidly during the first days of germination. The increase during the first 24 hours was almost as large when measured as specific activity (activity per mg protein in the mitochondrial fraction) than when measured on an absolute (i.e. per seed) basis; moreover, it was not significantly inhibited by puromycin or by actinomycin. The increase of the three activities during the following period of germination (second-third day) was accompanied by an increase of the protein nitrogen (per seed) in the mitochondrial fraction, and was consistently depressed by the protein synthesis inhibitors.

These results, integrated with those of other investigations on the same material are in agreement with the hypothesis that the activation of metabolism in the endosperm during germination depends in a very early phase mainly on the transition of enzyme systems from an inactive to an active state; while in a second phase synthesis « ex novo » of enzymes and cell structures predominates.     

Proteins in development and germination of a desiccation sensitive (recalcitrant) seed species

In the recalcitrant seeds of Avicennia marina, protein content and the rates of protein synthesis increase during histodifferentiation. This is similar to the situation in desiccation tolerant seeds. During the stage of reserve accumulation the protein content and rates of synthesis remain constant and there is no de novo synthesis of proteins which might qualify as storage proteins. There is also no change in the nature of proteins present in either axis or cotyledonary tissues during development or germination. Similarly, fluorographs of axis proteins show only very limited changes in the patterns of protein synthesis during development and germination, at least until the onset of root growth. Heat-stable proteins are present from an early developmental stage. However, no late embryogenic abundant (LEA) proteins are synthesised during the late stages of development, indicating that seedling establishment is independent of such maturation proteins. It is suggested that the lack of desiccation tolerance of A. marina seeds might be related to the absence of desiccation-related LEAs. Although the rate of protein synthesis increases during germination, protein metabolism appears to remain qualitatively the same as that occurring during development. The present results suggest that in these desiccation sensitive seeds, protein metabolism characterising development changes imperceptibly into that of germination.     

Protein and nucleic acid synthesis during germination of the asexual spores of the aquatic fungus, Aphanomyces astaci

Macromolecular synthesis of asexual spores of Aphanomyces astaci Schikora, strain is, was studied during the germination phase. Germination of the spores was completely inhibited by aclinomycin D (20 ug/ml) or cycloheximide (100 ug/ml) as was incorporation of labeled uridine and leucine, respectively. During spore germination protein synthesis was almost linear, whereas incorporation of [3Hl-uridine and [3H1-thymidine showed lag phases. Initial protein synthesis is therefore suggested to take place without concomitant RNA-synthesis in this species. Germlings were not developmentally competent to sporulate before 8 h of germination.     

Regulation of development of leucine uptake activity by glutamine in the scutellum of germinating barley grain

Scutella from ungerminated grains of barley (Hordeum vulgare L. cv Pirkka) take up leucine at a slow rate, which increases rapidly during germination. When endosperms were removed from the grains after imbibition for 4 hours or after germination for 12 or 72 hours, the increase in the rate of leucine uptake was greatly accelerated during subsequent incubation of the embryos or scutella. These increases were rapidly inhibited by cordycepin and cycloheximide, suggesting that protein synthesis, probably synthesis of the carrier protein, was required for the development of the uptake activity.

In separated embryos or scutella, the increases in the leucine uptake activity were inhibited by glutamine. The inhibitions caused by glutamine and cycloheximide were not additive, suggesting that glutamine did not interfere with the function of the carrier but repressed its synthesis. Glutamine did not inhibit the simultaneous increase in peptide uptake; in this respect, its effect was specific for leucine uptake, which appears to be due to a general amino acid uptake system.

Some other protein amino acids also inhibited the increase in leucine uptake without inhibiting the increase in peptide uptake. However, these effects were smaller than that of glutamine.

These results suggest that the transfer of leucine (and other amino acids) from the endosperm to the seedling in a germinating barley grain is regulated at the uptake step by repression of the synthesis of the amino acid carrier protein by glutamine and—possibly to a lesser extent—by some other amino acids taken up from the endosperm.

     

Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source

Activities of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.3) were measured in cotyledons of sunflower (Helianthus annuus L. cv Peredovic) seedlings during germination and early growth under various external nitrogen sources. The presence of NO ₃ ^- in the medium promoted a gradual increase in the levels of NR and NiR activities during the first 7 d of germination. Neither NR nor NiR activities were increased in a nitrogen-free medium or in media with either NH ₄ ⁺ or urea as nitrogen sources. Moreover, the presence of NH ₄ ⁺ did not abolish the NO ₃ ^- -dependent appearance of NR and NiR activities. The increase of NR activity was impaired both by cycloheximide and chloramphenicol, which indicates that both cytoplasmic 80S and plastidic 70S ribosomes are involved in the synthesis of the NR molecule. By contrast, the appearance of NiR activity was only inhibited by cycloheximide, indicating that NiR seems to be exclusively synthesized on the cytoplasmic 80S ribosomes. Glutamine-synthetase activity was also strongly increased by external NO ₃ ^- but not by NH ₄ ⁺ or urea. The appearance of GS activity was more efficiently suppressed by cycloheximide than chloramphenicol. This indicates that GS is mostly synthesized in the cytoplasm. The cotyledons of the dry seed contain high levels of GDH activity which decline during germination independently of the presence or absence of a nitrogen source. Cycloheximide, but not chloramphenicol, greatly prevented the decrease of GDH activity.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

Untersuchungen über rnasen in kotyledonen von nachgereiften und dormantenagrostemma githago-samen

Activities of RNasea were studied in cotyledons of dormant and afterripenedAgrostemma githago seeds. Activity of RNase increases during imbibition and germination. This increase in activity cannot be observed in variants which are not able to germinate (dormant seeds and seeds blocked by higher temperature). The development of RNase activities during germination cannot be inhibited by concentrations of cycloheximide or actinomycine D completely preventing phosphatase synthesis. These results may be indicative for the assumption that the increase of RNase during germination is caused by enzyme activation and not by enzyme synthesis. Cytokinins and a combination of cycloheximide and gibberellic acid stimulate the activity of RNase in dormant cotyledons, whereas neither cycloheximide nor gibberellic acid, applicated by themselves, show any effect. Cytokinins and gibberellic acid do not influence the activity of RNase of afterripened cotyledons, abscisic acid inhibits the increase of enzyme activity. There are characteristic changes in the pattern of RNases during germination revealed by polyacrylamide gel electrophoresis. The increase in RNase activity of dormant cotyledons caused by cytokinins is accompanied by obvious changes in the RNase pattern on polyacrylamide gel. Treating dormant cotyledons with cytokinins dormancy is partially overcome. In consequence of the application of cytokinins the differences in the electrophoretic RNase pattern between dormant and afterripened cotyledons can be nearly balanced.     

The glycolate pathway and photosynthetic competence in euglena

The development of glycolate pathway enzymes has been determined in relation to photosynthetic competence during the regreening of Euglena cultures. Phosphoglycolate phosphatase and glycolate dehydrogenase rapidly reached maximal levels of activity but the complete development of ribulose 1,5-diphosphate carboxylase and concomitant photosynthetic carbon dioxide fixation were not attained until 72 hours of illumination. Specific inhibitors of protein synthesis showed that the formation of ribulose 1,5-diphosphate carboxylase in both division-synchronized and regreening cultures was prevented by both cycloheximide and d-threo-chloramphenicol, whereas phosphoglycolate phosphatase formation was only inhibited by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide. Since cycloheximide prevented ribulose diphosphate carboxylase synthesis and photosynthetic carbon dioxide fixation without affecting phosphoglycolate phosphatase synthesis during regreening, it was concluded that photosynthetic competence was not necessary for the development of the glycolate pathway enzymes. The inhibition of phosphoglycolate phosphatase synthesis by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide shows that the enzyme was synthesized exclusively on chloroplast ribosomes, whereas protein synthesis on both chloroplast and cytoplasmic ribosomes was required for the formation of ribulose 1,5-diphosphate carboxylase. Although light is required for the development of both Calvin cycle and glycolate pathway enzymes during regreening it is concluded that the two pathways are not coordinately regulated.     

Protein Synthesis in Dormant and Non-Dormant Cocklebur Seed Segments

Using the axial and cotyledonary segments of lower cocklebur (Xanthium pensylvanicum Wallr.) seeds, protein synthesis as shown by incorporation of radioactive leucine was examined in relation to their dormant status. During the first 9 h of water imbibition, the protein synthesis was higher in the dormant axes than in the non-dormant, after- ripened ones. When imbibed for more than 12 h non-dormant axes had a higher activity than dormant ones. This was also the case with the cotyledonary segments. Cyctoheximide, an inhibitor of protein synthesis, blocked protein synthesis in the axial tissue regardless of its dormant status, and thereby inhibited germination of the non-dormant seeds. In the dormant seeds, however, cycloheximide at 3 mM slightly stimulated germination without stimulating the C₂H₄ production. Based on these results, it is suggested that in cocklebur seeds there may be some proteinaceous system which is involved in the maintenance of dormancy.