Purine and pyrimidine metabolism during the partial drying treatment of white spruce (Picea glauca) somatic embryos

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of ¹⁴C-labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO₂ and β - ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination.     

Pyrimidine deoxyribonucleotide metabolism during maturation and germination of white spruce (Picea glauca) somatic embryos: metabolic fate of C-labelled cytidine, deoxycytidine and thymidine

Pyrimidine deoxyribonucleotide metabolism was investigated during maturation and germination of white spruce somatic embryos by following the metabolic fate of [2‐¹⁴C]cytidine, [2‐¹⁴C]deoxycytidine and [2‐¹⁴C]thymidine. The de-novo pathway of deoxyribonucleotides was estimated indirectly, by the ability of the tissue to incorporate cytidine into DNA after conversion to dCTP. The salvage pathway was estimated by the utilization of labelled cytidine, deoxycytidine and thymidine for synthesis of deoxyribonucleotides and nucleic acids. Utilization of cytidine for DNA synthesis, via the de novo pathway, was always lower than that observed for RNA throughout the course of the experiment. Incorporation of cytidine into RNA was found to occur either directly, after conversion to CTP, mediated by the enzymes cytidine kinase, nucleoside monophosphate kinase and nucleoside diphosphate kinase, or indirectly, after conversion to UTP via uridine and UMP. Active incorporation of uridine into RNA of white spruce-cultured cells was demonstrated previously. Salvage of deoxycytidine and thymidine was operative in maturing and germinating white spruce somatic embryos, as label from both compounds was recovered in nucleotides and DNA. However, the utilization of these precursors by the cells was different. Salvage of deoxycytidine was always higher than that observed for thymidine, which was extensively catabolized to CO₂ at all stages of embryo development.     

Nonstructural carbohydrates in dormant and afterripened wild oat caryopses

Nonstructural carbohydrates were determined in both embryo and endosperm of dormant (nongerminating) and afterripened (germinating) intact caryopses of wild oat ( Avena fatua L.). No changes in endosperm starch or soluble sugar were observed at the onset of germination (18 h). No changes in glucose, fructose, sucrose or starch within dormant or afterripened embryos correlated with onset of visual germination. In afterripened embryos, depletion of raffinose (18 h), stachyose (18 h) and galactose (24 h) was correlated with germination. In contrast, raffinose-family oligosaccharide levels in dormant embryos remained constant for 7 days following imbibition. Germination of isolated dormant embryos on 88 m M galactose-containing media was accompanied by decreased endogenous levels of raffinose and stachyose. Isolated embryos from dormant caryopses incorporated ¹⁴C from ¹⁴C-fructose into both raffinose and stachyose during 24 h of imbibition. In contrast, no ¹⁴C incorporation into stachyose was observed in embryos from afterripened caryopses. No ¹⁴C incorporation into raffinose was observed at 18 and 24 h. When in vitro activities of α galactosidase were measured, no temporal differences between dormant or afterripened caryopses were detected in either embryo or endosperm tissue. Although the mechanism associated with differences in utilization of raffinose and stachyose is yet unidentified, alterations in raffinose-family oligosaccharide metabolism in the embryo appear to be a unique prerequisite for afterripening-induced germination.     

Changs in pyrimidine nucleotide biosynthesis during germination of white spruce (picea glauca) somatic embryos

Summary Changes in pyrimidine metabolism were investigated in germinating white spruce somatic embryos by following the metabolic fate of [2-¹⁴C]uracil and [2-¹⁴C]uridine, intermediate metabolites of the salvage pathway and [6-¹⁴C]orotic acid, a central metabolite of the de novo. nucleotide biosynthesis. An active uridine salvage was found to be responsible for the enlargement of the nucleotide pool at the inception of germination. Uridine kinase, which catalyzes the conversion of uridine to uridine monophosphate (UMP), was found to be very active in partially dried embryos and during the early phases of imbibition. The contribution of uracil to the nucleotide pool was negligible since a large amount of radioactivity from [2-¹⁴C]uracil was recovered in degradation products. As germination progressed, the decline of the uridine salvage pathway was concomitant with an increase of the de novo biosynthetic pathway. The central enzyme of the de novo pathway, orotate phosphoribosyltransferase, showed increased activity and contributed to the larger amount of orotate being anabolized. These results suggest that although both the salvage and de novo pathways operate in germinating white spruce somatic embryos, their contribution to the enlargement of the nucleotide pool appears tightly regulated as germination progresses.     

Pyrimidine metabolism during somatic embryo development in white spruce (Picea glauca)

Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce (Picea glauca). The contribution of thede novo and the salvage pathways of pyrimidine biosynthesis to nucleotide and nucleic acid formation and the catabolism of pyrimidine was estimated by the exogenously supplied [6-¹⁴C]orotic acid, an intermediate of thede novo pathway, and with [2-¹⁴C]uridine and [2-¹⁴C]uracil, substrates of the salvage pathways. Thede novo pathway was very active throughout embryo development. More than 80 percnt; of [6-¹⁴C]orotic acid taken up by the tissue was utilized for nucleotide and nucleic acid synthesis in all stages of this process. The salvage pathways of uridine and uracil were also operative. Relatively high nucleic acid biosynthesis from uridine was observed, whereas the contribution of uracil salvage to the pyrimidine nucleotide and nucleic acid synthesis was extremely limited. A large proportion of uracil was degraded as ¹⁴CO₂, probably via β-ureidopropionate. Among the enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase was high during the initial phases of embryo development, after which it gradually declined. Uridine kinase, responsible for the salvage of uridine, showed an opposite pattern, since its activity increased as embryos developed. Low activities of uracil phosphoribosyltransferase and non-specific nucleoside phosphotransferase were also detected throughout the developmental period. These results suggest that the flux of thede novo and salvage pathways of pyrimidine nucleotide biosynthesisin vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measuredin vitro did not exactly correlate with the flux estimated by the radioactive precursors. Therefore, other fine control mechanisms, such as the fluctuation of levels of substrates and/or effectors may also participate to the real control of pyrimidine metabolism during white spruce somatic embryo development.     

Comparative studies on pyrimidine metabolism in excised cotyledons of Pinus radiata during shoot formation in vitro

Changes in the pattern of pyrimidine nucleotide metabolism were investigated in Pinus radiata cotyledons cultured under shoot-forming (SF; +N(6)-benzyladenine) and non-shoot-forming (NSF, -N(6)-benzyladenine) conditions, as well as in cotyledons unresponsive (OLD) to N(6)-benzyladenine. This was carried out by following the metabolic fate of externally supplied (14)C-labeled orotic acid, intermediate of the de novo pathway, and (14)C-labeled uridine and uracil, substrates of the salvage pathway. Nucleic acid synthesis was also investigated by following the metabolic fate of (14)C-labeled thymidine during shoot bud formation and development. The de novo synthesis of pyrimidine nucleotides was operative under both SF and NSF conditions, and the activity of orotate phosphoribosyltransferase (OPRT), a key enzyme of the de novo pathway, was higher in SF tissue. Utilization of both uridine and uracil for nucleotide and nucleic acid synthesis clearly indicated that the salvage pathway of pyrimidine metabolism is also operative during shoot organogenesis. In general, uridine was a better substrate for the synthesis of salvage products than uracil, possibly due to the higher activity of uridine kinase (UK), compared to uracil phosphoribosyltransferase (UPRT). Incorporation of uridine into the nucleic acid fraction of OLD cotyledons was lower than that observed for their responsive (day 0) counterparts. Similarly, uracil utilization for nucleic acid synthesis was lower in NSF cotyledons, compared to that observed for SF tissue after 10 days in culture. This difference was ascribed to higher UPRT activity measured in the latter. Thus, there was an apparent difference in the utilization of nucleotides derived from uracil and uridine for nucleotide synthesis. The increased ability to produce pyrimidine nucleotides via the salvage pathway during shoot bud formation may be required in support of nucleic acid synthesis occurring during the process. Studies on thymidine metabolism confirmed this notion.     

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 white spruce somatic embryos: II. Continual shoot meristem development during germination

Summary This study compares the development of shoot apical meristems of white spruce somatic and zygotic embryos during germination. In mature somatic embryos, the functional part of the shoot apical meristem was bi-layered. After partial drying, a normal shoot meristem was formed from these two cell layers during germination. Other cells within the meristem were vacuolated and separated by intercellular air spaces. In the absence of the partial drying treatment, somatic embryos enlarged in size primarily due to vacuolation of cells and the formation of large intercellular air spaces. A majority of these somatic embryos failed to form a functional shoot apical meristem. Compared with somatic embryos, the shoot apical meristem of a mature zygotic embryo was well organized with a densely cytoplasmic apical layer. The cells within the meristem were tightly packed. Judging from the cell profiles during germination, all cells within the meristem of the zygotic embryo took part in the formation of the vegetative shoot apical meristem.     

Changes in Nucleic Acid Fractions of Seed Components of Red Pine (Pinus resinosa Ait.) during Germination

Changes in nucleic acid fractions of Pinus resinosa during seed germination were studied. At various stages of seed germination, embryos and megagametophytes were surgically separated and nucleic acids were extracted separately by a phenol-method. Total nucleic acids were fractionated on single-layer methylated albumin kieselguhr (MAK) columns. Total nucleic acids in embryos increased significantly 2 days after seeds were moistened, whereas, in megagametophytes, total nucleic acids stayed almost at a constant level until they degenerated at the time of shedding. In embryos, ribosomal RNAs (rRNA) increased 2 days after seeds were sown, whereas soluble RNA (sRNA) increased at 3 days. By comparison, nucleic acid fractions of megagametophytes did not show any quantitative changes during germination, except that rRNA fractions decreased shortly before shedding of seed coats. In dormant embryos the proportion of DNA was high and the proportions of sRNA and rRNA were low, whereas in megagametophytes at dormancy the proportions were completely reversed. As seed germination progressed, proportions of nucleic acid fractions in embryos changed significantly. In megagametophytes, although proportions of individual fractions remained almost constant throughout the experimental period, incorporation of ³²P into sRNA and rRNA of megagametophytes indicated turnover of these fractions.     

Specific features of the development of Siberian stone pine megagametophytes and embryos in vitro

Seedlings were grown in vitro from fertilized eggs and immature embryos of the Siberian stone pine. Cultivation of megagametophytes on a hormone-containing Murashige-Skoog medium from the egg formation until the globular embryo stage made it possible to manipulate fertilization and embryogenesis. Immature embryos are the most promising for in vitro cultivation. Their maturation and germination proceed within seven days of cultivation. When zygotic embryos were cultivated, adventitious buds were formed from cells at the cotyledon base and tips. When adventitious buds were subcultivated on a medium containing benzylaminopurine and naphthylacetic acid, organogenic callus and shoots were formed. Thus, cultivation of megagametophytes and embryos of the Siberian stone pine led to the completion of embryogenesis and formation of viable of seedlings.     

Brassinolide-improved development of <Emphasis Type="Italic">Brassica napus</Emphasis> microspore-derived embryos is associated with increased activities of purine and pyrimidine salvage pathways

Cellular brassinolide (BL) levels regulate the development of Brassica napus microspore-derived embryos (MDEs). Synthesis and degradation of nucleotides were measured on developing MDEs treated with BL or brassinazole (BrZ), a biosynthetic inhibitor of BL. Purine metabolism was investigated by following the metabolic fate of ¹⁴C-labelled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of both salvage and degradation pathways. For pyrimidine, orotic acid, uridine and uracil were employed as markers for the de novo (orotic acid), salvage (uridine and uracil), and degradation (uracil) pathways. Our results indicate that utilization of adenine, adenosine, and uridine for nucleotides and nucleic acids increased significantly in BL-treated embryos at day 15 and remained high throughout the culture period. These metabolic changes were ascribed to the activities of the respective salvage enzymes: adenine phosphoribosyltransferase (EC 2.4.2.7), adenosine kinase (EC 2.7.1.20), and uridine kinase (EC 2.7.1.48), which were induced by BL applications. The BL promotion of salvage synthesis was accompanied by a reduction in the activities of the degradation pathways, suggesting the presence of competitive anabolic and catabolic mechanisms utilizing the labelled precursors. In BrZ-treated embryos, with depleted BL levels, the salvage activity of both purine and pyrimidine nucleotides was reduced and this was associated to structural abnormalities and poor embryonic performance. In these embryos, the activities of major salvage enzymes were consistently lower to those measured in their control (untreated) counterparts.     

De novo pyrimidine nucleotide synthesis in the preimplantation mouse embryo

Utilizing phosphonacetyl- -aspartate (PALA), the transition state analog which specifically inhibits aspartate carbamyl transferase, we have shown that the preimplantation mouse embryo in culture has a functioning de novo pyridmidine biosynthetic pathway. This pathway accounts for some of the carbon dioxide fixation into nucleic acids previously described. Inhibition of de novo pyrimidine nucleotide synthesis during 2-cell to 8-cell development does not prevent morula development, but does prevent blastocyst development in nearly all embryos. Inhibition of the morula to blastocyst transition is most likely caused by a diminished pyrimidine nucleotide pool. Both de novo and salvage pathways appear active from the 2-cell embryo through blastocyst formation.     

Changes in mRNA populations during loblolly pine (Pinus taeda) seed stratification, germination and post-germinative growth

Changes in the patterns of gene expression were examined during loblolly pine ( Pinus taeda L.) seed stratification, germination, and post-germinative growth. In both the megagametophyte and the embryo, DNA contents remained relatively constant at all stages examined. RNA contents, however, increased in both tissues following seed germination, particularly in the embryo where a 7-fold increase in the RNA content was observed 5 days after germination. Poly(A)⁺ RNA, extracted from megagametophytes and embryos, was translated in vitro in a rabbit reticulocyte lysate cell-free system. Analysis of [³⁵S]-methionine-labelled translation products by two-dimensional electrophoresis/fluorography indicated that there were changes in the populations of mRNAs during all developmental stages examined. In both the megagametophyte and the embryo several distinct mRNA populations, including one constitutively present at all stages examined, were identified. One mRNA population, present in the mature seed, decreased during seed stratification. Another population, not present in the mature seed, rose during the period of stratification that coincided with an increase in seed germinability. A third population, which appeared during seed germination, increased steadily during post-germinative growth. Besides these similarities, specific differences between megagametophyte and embryo were noted. For example, one mRNA population, which was present in the megagametophyte of the mature seed and remained constant during the stratification period, disappeared immediately following seed germination. In the embryo, one set of messages was germination specific. In total, these results show that mRNA populations change in a temporal fashion that is consistent with the patterns of de novo protein synthesis known to occur in loblolly pine during the same developmental periods.     

Specific Features of Development of Megagametophytes and Embryos of the Siberian Stone Pine in vitro

Seedlings were grown in vitro from fertilized eggs and immature embryos of the Siberian stone pine. Cultivation of megagametophytes on a hormone-containing Murashige-Skoog medium from the egg formation until the globular embryo stage made it possible to manipulate fertilization and embryogenesis. Immature embryos are the most promising for in vitrocultivation. Their maturation and germination proceed within seven days of cultivation. When zygotic embryos were cultivated, adventitious buds were formed from cells at the cotyledon base and tips. When adventitious buds were subcultivated on a medium containing benzylaminopurine and naphthylacetic acid, organogenic callus and shoots were formed. Thus, cultivation of megagametophytes and embryos of the Siberian stone pine led to the completion of embryogenesis and formation of viable of seedlings.     

Pyridine nucleotide cycle and trigonelline (N-methylnicotinic acid) synthesis in developing leaves and fruits of Coffea arabica

We examined the biosynthesis of trigonelline in leaves and fruits of Arabica coffee ( Coffea arabica ) plants. [³H]Quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [¹⁴C]nicotinamide and [¹⁴C]nicotinic acid, which are degradation products of NAD, were converted to trigonelline and pyridine nucleotides. These tracer experiments suggest that the pyridine nucleotide cycle, nicotinamide → nicotinic acid → nicotinic acid mononucleotide (NaMN) → nicotinic acid adenine dinucleotide (NaAD) → NAD → nicotinamide mononucleotide (NMN) → nicotinamide, operates in coffee plants, and trigonelline is synthesized from nicotinic acid formed in the cycle. Trigonelline accumulated up to 18 µmol per leaf in developed young leaves, and then decreased with age. Although the biosynthetic activity of trigonelline from exogenously supplied [¹⁴C]nicotinamide was observed in aged leaves, the endogenous supply of nicotinamide may be limited, reducing the contents in these leaves. Trigonelline is synthesized and accumulated in fruits during development. The trigonelline synthesis in pericarps is much higher than that in seeds, but its content in seeds is higher than pericaps, so that some of the trigonelline synthesized in the pericarps may be transported to seeds. Trigonelline in seeds may be utilized during germination, as its content decreases. Trigonelline synthesis from [¹⁴C]nicotinamide was also found in Theobroma cacao plants, but instead of trigonelline, nicotinic acid-glucoside was synthesized from [¹⁴C]nicotinamide in Camellia sinensis plants.     

Histones and the first cell cycle in maize germination

The timing of the onset of cell division during seed germination in maize and the role of histones for this process have been studied. Embryonic axes of maize seeds ( Zea mays L. hybrid H-30) were incubated in a sterile nutrient medium for different periods of time. For some experiments putrescine was also added. Mesocotyl, root tip and scutellar node were dissected at specific periods after incubation and the mitotic indices were determined in these tissues. Embryonic axes were incubated in the same medium either with [¹⁴C]-lysine or [³²P]-phosphate. The incorporation of either ¹⁴C or ³²P into histones was followed, both in postribosomal supernatant and in nuclei. It was found that during germination, there is specific timing for meristematic cells entering into cell division. Among the tissues tested, the mesocotyl meristem was the first to initiate this process. De novo synthesis of histones was detected as early as after 6 h of imbibition and the rate increased up to 12 h. Putrescine stimulated cell division and phosphorylation of the histones. The implications of these findings are discussed.     

Cell Position Dependence of Labelling Thymidine Nucleotides Using the De Novo and Salvage Pathways In the Crypt of Small Intestine

Abstract. About twice as much tritiated thymidine ([³H]TdR) is taken up by cells at the bottom of the crypt of the small intestine as by the rapidly cycling mid-crypt cells. However, the uptake of tritiated deoxyuridine ([³H]UdR) is even throughout the crypt.
Exogenous thymidine is incorporated about four times and eight times more efficiently than deoxyuridine by the cells in the mid-crypt and cells at the bottom of the crypt, respectively. However all S phase cells in the crypt appear to be capable of using either precursors, i.e. either the de novo or salvage pathway.
Since methotrexate (1 or 5 mg/kg) inhibits (at 5 mg/kg completely) the uptake of [³H]UdR, but has no effect on [³H]TdR uptake, the de novo and salvage pathways appear to be independent. Within the precision of the methods used in the experiments the 3 hr inhibition of the de novo pathway of deoxythymidylic acid (dTMP) synthesis by methotrexate does not produce any increase in utilization of the salvage pathway measured by incorporation of [³H]TdR into DNA. the increased efficiency of thymidine utilization by crypt base cells is not attributable to (i) differences in accessibility of thymidine; (ii) differences in the rate of DNA synthesis or (iii) the size of the nuclei.     

Purine Nucleotide Synthesis in Adrenal Chromaffin Cells

Abstract: The synthesis of purine nucleotides from the salvage precursors adenine and adenosine, and from the de novo precursors formate and glycine, was studied in isolated adrenal chromaffin cells. Both [8-¹⁴C]adenine and [8-¹⁴C]adenosine from extracellular medium are effectively incorporated into intracellular nucleotides. [¹⁴C]Formate and [U-¹⁴C]glycine are also incorporated, but de novo synthesis is clearly lower than synthesis from salvage precursors, although similar to de novo synthesis in liver. The enzymes responsible for adenine and adenosine salvage, adenine phosphoribosyltransferase and adenosine kinase, were purified about 1,500-fold. Both enzymes are mainly cytosolic and exhibit a similar molecular weight of around 42,000. The results suggest that chromaffin cells can replenish their intracellular nucleotides lost during the secretory event by an active synthesis from salvage and de novo precursors.     

Progression of programmed cell death in tobacco BY-2 cells is delineated by specific changes in de novo and salvage synthesis of purine nucleotides

Alterations in the pattern of purine nucleotide synthesis and degradation were investigated during programmed cell death (PCD) of tobacco BY-2 cells, induced by a simultaneous increase in the endogenous levels of nitric oxide (NO) and hydrogen peroxide. The de novo synthesis of purine nucleotides was estimated by following the metabolic fate of the [8-¹⁴C]5-aminoimidazole-4-carboxamide-1-β- d -ribofuranoside (AICAR), the salvage synthesis was investigated using [8-¹⁴C]adenine and adenosine, and the degradation pathway was studied by following the incorporation of [8-¹⁴C]inosine. The results indicated that specific changes in purine metabolism occurred during the death programme of tobacco cells. During the early phases of PCD, increases in the salvage activity of adenine and adenosine were observed, and these were related to the high activity of the two major salvage enzymes: adenine phosphoribosyltransferase (APRT) and adenosine kinase (ARK). During the following stages, a large fraction of purine nucleotide was also produced through the de novo pathway, suggesting a tight regulation between salvage and de novo synthesis. These changes were strictly associated with PCD, as they did not occur if NO or hydrogen peroxide was increased individually, or if actinomycin, which inhibits the death programme, was added to the medium in the presence of NO and hydrogen peroxide. These changes in purine nucleotide synthesis represent an early metabolic switch which may be needed to ensure the proper execution of all the high-energy demand processes characteristic of the death programme.