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.     

The development of ribonuclease and acid phosphatase during germination of Pisum arvense

1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.     

Polymorphism in rice amylases at an early stage of seed germination

A polymorphism in rice amylases at an early stage of seed germination is analyzed by zymogram. In non-glutinous cultivars of rice, alpha-amylase isozymes are mainly confirmed in germinating seeds. However, in glutinous cultivars, beta-amylase isozymes, which are not confirmed in nonglutinous cultivars, make up the major part of the total amylase activity and the expression of alpha-amylases are repressed.     

Purification and some properties of a Haim-sensitive alpha-amylase from newly isolated Bacillus sp. No. 195.

Newly isolated Bacillus sp. No. 195 produced an extracellular alpha-amylase sensitive to Haim which was found to inhibit specifically animal alpha-amylases. The enzyme was purified easily by two steps of starch adsorption and gel filtration using Sephacryl S-200. The purified enzyme, which showed a single band on native-PAGE or SDS-PAGE, had a molecular weight of 60,000 as judged on SDS-PAGE. The optimum pH value for activity and the isoelectric point were around 7.0 and 4.5, respectively. The sensitivity of the amylase to Haim was similar to that of animal amylase rather than bacterial amylase. It was suggested that a Haim-amylase complex might be formed at the molar ratio of 1:1. The amino acid sequence F-S-W similar to the triplet F-E-W highly conserved among alpha-amylases sensitive to proteinaceous inhibitors, such as Hoe 467-A or Haim, was found in the amino-terminal part of the No. 195 amylase.     

Effect of histidine on purine nucleotide synthesis and utilization in Neurospora crassa.

Histidine affects de novo purine nucleotide synthesis and purine nucleotide pool utilization in Neurospora crassa. The former effect was assessed qualitatively by the presence or absence of purple pigment production in ad3B and ad3A mutants. Tryptophan also affected the de novo purine nucleotide synthesis. The effect of histidine on purine nucleotide pool utilization resulted in stimulated germination of ad8 and ad4 mutant conidia in adenine-deficient medium. Increased germination was correlated with increased net levels of nucleic acids in these strains. Possible mechanisms for the dual action of histidine are discussed.     

Ribonucleic acid polymerase of germinating Bacillus cereus T.

It appears that a de novo synthesis of the deoxyribonucleic acid-dependent ribonucleic acid-polymerase in Bacillus cereus T takes place fairly late in outgrowth, at the onset of the vegetative cycle. Therefore, the ribonucleic acid-polymerase used by germinating spores is the one carried on from sporulating cells. However, the sporal enzyme is less soluble that the vegetative one, and its "core" is bound to two extra peptides. This complexing to other molecules could play a role in the regulation of gene expression during germination.     

A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other alpha-amylases, such as Taka-amylase A. The 48-kilodalton (kDa) amylase isolated from B. polymyxa was proven to have alpha-amylase activity. The amino acid sequences of the peptides generated from the 48-kDa amylase showed complete agreement with the predicted amino acid sequence of the C-terminal portion. The B. polymyxa amylase gene was therefore concluded to contain in-phase beta- and alpha-amylase-coding sequences in the 5' and 3' regions, respectively. A precursor protein, a 130-kDa amylase, directed by a plasmid, pYN520, carrying the entire amylase gene, had both beta- and alpha-amylase activities. This represents the first report of a single protein precursor in procaryotes that gives rise to two enzymes.     

Alcohol dehydrogenase in Arabidopsis: analysis of the induction phenomenon in plantlets and tissue cultures

Summary Alcohol dehydrogenase (ADH) activity is expressed in Arabidopsis seeds and tissue cultures. During the germination process, ADH activity declines rapidly and is no longer detectable in 9- to 10-day-old seedlings. The synthesis of ADH could be demonstrated in seedlings submitted to anaerobiosis by ³⁵S-methionine incorporation studies.Callus, induced from seeds or leaves on a 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, and cell suspension cultures are characterized by a high level of ADH activity. The incorporation of ³⁵S-methionine and two-dimensional electrophoresis indicated that ADH induction was due to de novo synthesis of the polypeptides. In vitro translation of total poly (A)⁺-RNA from seedlings and callus showed that only callus mRNA was able to direct the synthesis of ADH polypeptides. This demonstrates the de novo synthesis of ADH mRNA during callus induction.Northern blot hybridization, using in vitro labelled ADH1-F DNA from maize as a probe, revealed sequence homology at the mRNA level between Arabidopsis and maize.Dedicated to professor Georg Melchers to celebrate his 50-year association with the journal     

The cloning and characterization of a second alpha-amylase of A. hydrophila JMP636

AIMS: The aim of this study was to identify, clone and characterize the second amylase of Aeromonas hydrophila JMP636, AmyB, and to compare it to AmyA.METHODS AND RESULTS: The amylase activity of A. hydrophila JMP636 is encoded by multiple genes. A second genetically distinct amylase gene, amyB, has been cloned and expressed from its own promoter in Escherichia coli. AmyB is a large alpha-amylase of 668 amino acids. Outside the conserved domains of alpha-amylases there is limited sequence relationship between the two alpha-amylases of A. hydrophila JMP636 AmyA and AmyB. Significant (80%) similarity exists between amyB and an alpha-amylase of A. hydrophila strain MCC-1. Differences in either the functional properties or activity under different environmental conditions as possible explanations for multiple copies of amylases in JMP636 is less likely after an examination of several physical properties, with each of the properties being very similar for both enzymes (optimal pH and temperature, heat instability). However the reaction end products and substrate specificity did vary enough to give a possible reason for the two enzymes being present. Both enzymes were confirmed to be alpha-type amylases.CONCLUSIONS: AmyB has been isolated, characterized and then compared to AmyA.SIGNIFICANCE AND IMPACT OF THE STUDY: The amylase phenotype is rarely encoded by more than one enzyme within one strain, this study therefore allows the better understanding of the unusual amylase production by A. hydrophila.     

Exogenous gibberellins inhibit coffee (Coffea arabica cv. Rubi) seed germination and cause cell death in the embryo

The mechanism of inhibition of coffee (Coffea arabica cv. Rubi) seed germination by exogenous gibberellins (GAs) and the requirement of germination for endogenous GA were studied. Exogenous GA(4+7) inhibited coffee seed germination. The response to GA(4+7) showed two sensitivity thresholds: a lower one between 0 and 1 microM and a higher one between 10 and 100 microM. However, radicle protrusion in coffee seed depended on the de novo synthesis of GAs. Endogenous GAs were required for embryo cell elongation and endosperm cap weakening. Incubation of coffee seed in exogenous GA(4+7) led to loss of embryo viability and dead cells were observed by low temperature scanning microscopy only when the endosperm was surrounding the embryo. The results described here indicate that the inhibition of germination by exogenous GAs is caused by factors that are released from the endosperm during or after its weakening, causing cell death in the embryo and leading to inhibition of radicle protrusion.     

Changes in the de novo, salvage, and degradation pathways of pyrimidine nucleotides during tobacco shoot organogenesis.

Pyrimidine nucleotide metabolism was studied in tobacco callus cultured for 21days under shoot-forming (SF) and non-shoot-forming (NSF) conditions by following the metabolic fate of orotic acid, a precursor of the de novo pathway, and uridine and uracil, intermediates of the salvage and degradation pathways respectively. Nucleic acid synthesis was also investigated by measuring the incorporation of labeled thymidine into different cellular components. Our results indicate that with respect to nucleotide metabolism, the organogenic process in tobacco can be divided in two "metabolic phases": a de novo phase followed by a salvage phase. The initial stages of meristemoid formation during tobacco organogenesis (up to day 8) are characterized by a heavy utilization of orotic acid into nucleotides and nucleic acids. Utilization of this intermediate for the de novo synthesis of nucleotides, which is limited in NSF tissue, is mainly due to the activity of orotate phosphoribosyltransferase (OPRT), which increases in tissue cultured under SF conditions. After day 8, nucleotide synthesis during shoot growth seems to be mainly due to the salvage activity of both uridine and uracil. Both intermediates are preferentially utilized in SF tissue for the formation of nucleotides and nucleic acids through the activities of their respective salvage enzymes: uridine kinase (URK), and uracil phosphoribosyltransferase (UPRT). Metabolic studies on thymidine indicate that in SF tissue maximal nucleic acid synthesis occurs at day 4, in support of the initiation of meristemoid formation. Overall these results suggest that the organogenic process in tobacco is underlined by precise fluctuations in pyrimidine metabolism which delineate structural events culminating in shoot formation.     

Development of RNase activity in embryonic axes of germinating pea seeds

RNase activity in embryonic pea axes increased in parallel withthe rise of RNA synthesis as germination proceeded. The developmentof this enzymatic activity was modified antagonistically byapplication of GA₃ and ABA and inhibited severely by treatmentwith CH. Sedimentation analysis of ³H-adenosine-labeled RNAindicated that the synthesis of all types of RNA species isuniformly stimulated by GA₃ and inhibited by ABA. However, 5-FUtreatments, which severely inhibited the synthesis of rRNA,with a slight effect on that of mRNA, had no appreciable effecton the development of RNase activity in the axes. These resultsindicate that active RNA synthesis during germination is independentof the development of RNase activity and that the de novo synthesisof RNases may be controlled by the synthesis of their specificmRNAs. Among the three types of RNase (RNase I, II and III) detectedin the embryonic axes, RNase III showed a sharp increase inactivity with embryo growth and the activity of this enzymewas mainly associated with the endoplasmic reticulum. (Received June 5, 1978; )     

Die zeitliche Dauer der Isocitrat-Lyase-Synthese in Kotyledonen von Wassermelonenkeimlingen

Summary Previously, it was deduced from inhibitor experiments that isocitrate lyase (EC 4.1.3.1.) is synthesized de novo in watermelon cotyledons during the first 3 days of germination, which explains the sharp increase of activity during this period. The following decrease of activity was interpreted as the result of a limited half life of the enzyme molecule (Hock and Beevers, 1966).This hypothesis has been confirmed now by density labeling experiments of isocitrate lyase with deuterium. Seedlings grown from day 0 on D₂O (80 vol. %) contained a heavier enzyme at the time of maximum activity than control seedlings grown on H₂O (Fig. 6). No incorporation of deuterium into isocitrate lyase, however, was detectable when the cotyledons were labeled only from day 3 1/2 on, i.e. after the stage of maximum activity had been passed (Fig. 10), in spite of the fact that D₂O was taken up from the cotyledons in considerable quantities. —These results prove at the same time that density labeling of the isocitrate lyase during early stages of germination was a result of de novo synthesis rather than a mere artifact produced by isotopic exchange.An improved method for the purification of isocitrate lyase from higher plants is introduced.     

Proteases involved in generation of beta- and alpha-amylases from a large amylase precursor in Bacillus polymyxa.

The genes for extracellular neutral protease (Npr) and intracellular serine protease (Isp) were cloned from Bacillus polymyxa in order to elucidate the process involved in the generation of multiple beta-amylases and an alpha-amylase from a large amylase precursor. The npr gene was composed of 1,770 bp and 570 amino acids, while the isp gene was composed of 978 bp and 326 amino acids. Both proteases produced by E. coli cleaved the amylase precursor to generate beta- and alpha-amylases. Furthermore, several other proteases produced the same products from the precursor. A 130-kDa amylase precursor has two large domain structures responsible for the generation of beta- and alpha-amylases. The junction region of approximately 200 amino acids may be exposed on the surface of the molecule and susceptible to proteolytic enzymes, which results in the formation of multiple amylases.     

Reactive products formed by peroxidase catalyzed oxidation of p-phenetidine

The drug 4-nitroquinoline 1-oxide (4NQO) is a potent inhibitor of Dictyostelium discoideum spore germination. This inexpensive, water soluble drug is active at a concentration of 5 micrograms/ml (26 microM) and permeates the spore at all stages in germination. Spores subjected to 4NQO treatment exhibit an irreversible blockage of myxamoebae emergence, but spore activation, post-activation lag, and swelling are not affected. Swollen 4NQO-treated spores lose the outer two spore walls but lack the ability to degrade the innermost wall. The drug does not affect oxygen uptake during post-activation lag or swelling, and only a stage specific depression in O2 uptake is observed when control spores begin to release myxamoebae. When added early in germination, 4NQO blocks the incorporation of [3H] uracil into a cold trichloroacetic acid (TCA) insoluble fraction by 98%. However, when the drug is added midway through germination and followed by a pulse labelling period of 1 h, only 65% inhibition of RNA synthesis is observed. This lack of complete inhibition may occur because the drug requires metabolic activation; thus, new rounds of RNA synthesis may have initiated before the drug became fully activated. 4NQO also blocks the de novo expression of beta-glucosidase activity when added early in germination. Additionally, we observe that vegetative cellular slime mold cells are 100 times more resistant than spores to 4NQO-induced damage. Taken together, our results support the observation that RNA synthesis is only required for the emergence stage of germination and that dormant D. discoideum spores may lack efficient excision repair mechanisms.