White Spruce Somatic Embryos Research Articles

Effects of exogenous polyamines and inhibitors of polyamine biosynthesis on endogenous free polyamine contents and the maturation of white spruce somatic embryos

The maturation of somatic embryos is a pivotal stage of somatic embryogenesis process. Thus, this study is aimed at investigating the role of polyamines on the maturation of somatic embryos of white spruce (Picea glauca). Two inhibitors of the biosynthesis of polyamines methylglyoxal bis-(guanylhydrazone) (MGBG) and dicyclohexylamine (DCHA) or three exogenous polyamines (putrescine, spermidine and spermine) were added into a modified HLM-1 maturation medium inoculated with embryogenic tissues. Medium responses were assessed with respect to the production of mature somatic embryos, the cellular concentration of free polyamines and the concentration of 1- aminocyclopropane-1-carboxylic acid (ACC) and N-malonylaminocyclopropane-1-carboxylic acid (mACC), the precursor of ethylene biosynthesis and its conjugate, respectively. Results show that DCHA brought about a significant decrease in the concentration of free cellular putrescine during the first two weeks of maturation, whereas MGBG induced a significant decrease of both spermidine and spermine. Moreover, both inhibitors (1 mM DCHA or 1 mM MGBG) reduced the total number (63 and 52%, respectively) and the number of normal (62 and 54%, respectively) somatic embryos. Addition of 5 mM spermidine or 1 mM spermine increased the total number (94 and 113%, respectively) and the number of normal (134 and 143%, respectively) somatic embryos. Finally, the intracellular concentrations of ACC and mACC significantly increased in the presence of exogenous spermidine while decreasing in the presence of MGBG, suggesting that spermidine interacts with ethylene metabolism. These results clearly demonstrate that polyamines play an important quantitative and qualitative role during the maturation of white spruce somatic embryos.

Cellular ascorbic acid regulates the activity of major peroxidases in the apical poles of germinating white spruce (Picea glauca) somatic embryos

In previous studies we have reported that applications of ascorbic acid (ACS) enhance the conversion frequency of white spruce somatic embryos by “rescuing” structurally disorganized meristems and inducing cell proliferation in the apical poles [C. Stasolla, E.C. Yeung, Ascorbic acid improves the conversion of white spruce somatic embryos, In Vitro Cell. Dev. Biol. Plant 35 (1999) 316–319]. In order to determine if the role played by this metabolite during embryo conversion is mediated by cellular peroxidases, the activity of guaiacol-, ferulic acid-, and ascorbic acid-dependent peroxidases were measured in the apical poles of germinating embryos with altered ASC levels. Changes in the endogenous ASC pool were achieved by treating the embryos with exogenously supplied ASC, l-galactono-γ-lactone (GL) the last precursor of the de novo biosynthesis of ASC, and lycorine (L), an inhibitor of the last reaction leading to the synthesis of ASC. Our studies demonstrate the existence of a negative correlation between cellular ASC levels and activities of both guaiacol and ferulic acid peroxidases in root and shoot apices. A depletion of cellular ASC enhanced the rate of both guaiacol and ferulic acid oxidation at the apical poles of the embryos and resulted in meristem abortion. In contrast, the activity of guaiacol and ferulic acid peroxidases decreased below control levels if the endogenous ASC content of the embryos was experimentally increased. Fluctuations of total peroxidase activity following alterations in ASC pool were also confirmed by histochemical staining and in vitro studies. Overall our results suggest that a threshold of ASC level must be maintained in the apical poles of germinating embryos in order to inhibit peroxidase activities from cross-linking cell wall components and preventing post-embryonic growth.

Aminocyclopropane Carboxylic Acid Synthase Is a Regulated Step in Ethylene-Dependent Induced Conifer Defense. Full-Length cDNA Cloning of a Multigene Family, Differential Constitutive, and Wound- and Insect-Induced Expression, and Cellular and Subcellular Localization in Spruce and Douglas Fir

In conifer stems, formation of chemical defenses against insects or pathogens involves specialized anatomical structures of the phloem and xylem. Oleoresin terpenoids are formed in resin duct epithelial cells and phenolics accumulate in polyphenolic parenchyma cells. Ethylene signaling has been implicated in the induction of these chemical defenses. Recently, we reported the cloning of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) from spruce (Picea spp.) and Douglas fir (Pseudotsuga menziesii). ACO protein was constitutively expressed in Douglas fir and only weakly induced upon wounding. We now cloned seven full-length and one near full-length cDNA representing four distinct 1-aminocyclopropane-1-carboxylic acid synthases (ACS; ACS1, ACS2, ACS3, and ACS4) from spruce and Douglas fir. Cloning of ACS has not previously been reported for any gymnosperm. Using gene-specific, quantitative real-time polymerase chain reaction, we measured constitutive expression for the four ACS genes and the single-copy ACO gene in various tissues of Sitka spruce (Picea sitchensis) and in white spruce (Picea glauca) somatic embryos. ACO and ACS4 were ubiquitously expressed at high levels; ACS1 was predominantly expressed in developing embryos and ACS2 and ACS3 were expressed only at very low levels. Insect attack or mechanical wounding caused strong induction of ACS2 and ACS3 in Sitka spruce bark, a moderate increase in ACO transcripts, but had no effect on ACS1 and ACS4. ACS protein was also strongly induced following mechanical wounding in Douglas fir and was highly abundant in resin duct epithelial cells and polyphenolic parenchyma cells. These results suggest that ACS, but not ACO, is a regulated step in ethylene-induced conifer defense.

Endogenous ascorbic acid modulates meristem reactivation in white spruce somatic embryos and affects thymidine and uridine metabolism

Previously, we demonstrated that, in some cell lines, exogenous applications of ascorbic acid (ASC) enhance the conversion frequency of white spruce (Picea glauca (Moench) Voss) somatic embryos, by stimulating mitotic activity in the apical meristems. To examine this event in more detail, we investigated the effects of ASC on de novo, salvage and degradation pathways of pyrimidine metabolism by following the metabolic fate of (14)C-labeled orotic acid, thymidine, uridine and uracil in shoot and root poles of germinating embryos, after altering the cellular ASC content of the embryos. Alterations in endogenous ASC content did not affect the utilization of either orotic acid or uracil, but affected the metabolism of thymidine and uridine. Specifically, a lowering of endogenous ASC content by applications of lycorine (L), an inhibitor of the last enzyme of the ASC de novo biosynthetic pathway, resulted in a lower embryo conversion frequency, as well as a reduced percentage of thymidine and uridine incorporated into nucleotides and nucleic acids. The reduction in thymidine and uridine anabolism was mainly ascribed to the decreased activities of thymidine kinase (TRK) and uridine kinase (URK), the respective salvage enzymes of thymidine and uridine, measured in L-treated embryos. These effects were solely a result of a decrease in endogenous ASC content because applications of ascorbic acid plus lycorine (ASC + L) increased embryo conversion frequency, thymidine and uridine salvage activities, and TRK and URK activities to near control values. Inclusion of exogenous ASC in the germination medium did not affect the percentage of embryos able to convert to viable plantlets, although it increased thymidine and uridine utilization for nucleic acid synthesis in the shoot and root poles of the embryos. Taken together, these findings confirm that cellular ASC plays a key role in the reactivation of the apical meristems of germinating white spruce somatic embryos.

Exogenous Applications of Ascorbic Acid Enhance Shoot Apical Meristem Growth and Induce Shoot Organogenesis in Germinating White Spruce (Picea glauca) Somatic Embryos

Ascorbic acid (ASC) has been shown to improve the postembryonic growth of white spruce somatic embryos. In order to comprehend the effects of ASC applications on shoot apical meristem reactivation, structural studies were conducted. In control embryos, postembryonic growth was dependent on the quality of the shoot apical meristems since only well‐organized meristems were able to resume mitotic activity at germination and undergo successful conversion. Resumption of cell division, in fact, was seldom observed in those embryos with poorly organized shoot meristems. ASC was found to rescue these structurally disorganized meristems by stimulating cell division in the apical pole. The newly divided cells were able to develop further and to generate a functional shoot primordium through the process of organogenesis. Mitotic divisions were also observed at the base of the cotyledons; this often resulted in the formation of an additional shoot apical meristem, especially if the main apex failed to develop.

The effect of osmoticum on ascorbate and glutathione metabolism during white spruce ( Picea glauca) somatic embryo development

Water stress is an important factor which regulates organized development of both zygotic and somatic embryos. Somatic embryos of white spruce were cultured in the presence of polyethylene glycol (PEG), a non-plasmolyzing agent which increases embryo quality and number, and mannitol, a plasmolyzing agent. The effects of these two compounds on both ascorbate and glutathione metabolism were investigated at different stages of embryo development. Compared to control and mannitol-treated embryos, embryos treated with PEG accumulated higher levels of endogenous ascorbate (ASC) in its reduced form, especially during the first half of the maturation period. This increase, also observed in immature seeds, was mainly the result of two different processes: activation of the de novo ASC machinery, and recycling of ASC from ascorbate free radicals (AFR) which was modulated by the activity of ascorbate free radical reductase (AFRR, EC. 1.6.5.4). The activity of this enzyme increased during the early phases of development in both PEG-treated somatic embryos and seeds. Compared to control somatic embryos, mannitol and PEG were shown to change the levels of reduced (GSH) and oxidized glutathione (GSSG). In particular, a constant decline in the GSH/GSSG ratio was observed in the presence of PEG. This pattern was also observed in maturing white spruce seeds. Overall, these data indicate that applications of non-plasmolyzing agents in the culture medium of spruce somatic embryos result in seed-like fluctuations of the ascorbate–glutathione metabolism, which may have a positive effect on embryo yield.

The effect of reduced glutathione on morphology and gene expression of white spruce (Picea glauca) somatic embryos.

Inclusions of reduced glutathione (GSH) in the maturation medium increased the conversion frequency of white spruce somatic embryos without the need of a partial drying treatment (PDT). This beneficial effect was the result of major alterations in morphology and gene expression during the maturation period. Compared with control embryos, GSH-treated embryos showed a differential accumulation of storage products, i.e. preferential deposition of starch, the reduced formation of protein bodies, and increased vacuolation of cells. These morphological changes correlated with extensive alterations of gene expression occurring throughout the maturation period. The transcript profiles of stage-specific embryos matured with or without GSH were analysed using a DNA microarray containing 2 178 cDNAs from loblolly pine (Pinus taeda). The efficiency of heterologous hybridization between spruce and pine species on microarrays has previously been documented. The results indicate that several genes involved in a variety of signal regulatory pathways were differentially expressed in developing GSH- treated embryos. The transcript levels of many genes involved in carbohydrate metabolism and protein synthesis were altered by the presence of GSH and denoted differences in physiology between treatments. Extensive changes in the expression of genes participating in hormone synthesis, nucleotide metabolism, and meristem formation were also observed and related to the post-embryonic performance of the embryos.

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.

Transcript profiles of stress-related genes in developing white spruce ( Picea glauca) somatic embryos cultured with polyethylene glycol

The effect of polyethylene glycol (PEG) on the transcript level of 512 stress-related genes was analyzed by cDNA microarray. Major changes in gene expression between control and PEG-treated embryos were observed during the initial stages of development, upon transfer of the embryogenic tissue on maturation medium, and during the late phases of development, culminating with the generation of cotyledonary embryos. Only small changes in gene expression were observed during the intermediate phases of embryo development. The transcript levels of several genes involved in cell aging and detoxification mechanisms, including peroxidases and chitinases, were developmentally regulated during the embryogenic process. Major differences in the expression of these genes were observed between control and PEG-treated embryos. Based on their expression profiles, four different clusters of genes involved in stress response mechanisms were identified. The first group of genes, which included several heat shock proteins, was up-regulated in PEG-treated immature embryos. An opposite tendency was observed for a second cluster of genes, which included a glutathione-S-transferase, and a cysteine protease. The third class included genes repressed by PEG in fully developed embryos, whereas a fourth group of genes, which included several heat shock proteins and ubiquitin, was induced in PEG-treated embryos at the end of the culture period. Difference in transcript levels and profiles of several genes involved in cell wall and lignin biosynthesis were also observed between control and PEG-treated embryos.

The Effects of Polyethylene Glycol on Gene Expression of Developing White Spruce Somatic Embryos

Somatic embryogenic cultures of white spruce (Picea glauca) represent a valuable system to study molecular mechanisms regulating embryo development because many embryos of defined developmental stages can be generated. The inclusion of polyethylene glycol (PEG) in the maturation medium can improve the number and quality of embryos produced. To learn more about the mechanism of action of PEG, we analyzed transcript profiles of stage-specific embryos matured without (control) or with (PEG treated) PEG. RNA extracted from maturing spruce embryos was analyzed on DNA microarrays containing 2,178 cDNAs from loblolly pine (Pinus taeda). The efficiency of heterologous hybridization between spruce and pine species on microarrays has been documented previously (L. van Zyl, S. von Arnold, P. Bozhkov, Y. Chen, U. Egertsdotter, J. MacKay, R. Sederoff, J. Shen, L. Zelena, D. Clapham [2002] Comp Funct Genomics 3: 306-318). Several pine genes, including the apparent homologs to the Arabidopsis genes ZWILLE, FIDDLEHEAD, FUSCA, and SCARECROW, increased in expression after PEG treatments. These genes are known to be involved in the formation of the embryo body plan and in the control of the shoot and root apical meristems. The increased transcript levels of these genes in immature PEG-treated embryos suggest that PEG may improve the quality of spruce somatic embryos by promoting normal differentiation of the embryonic shoot and root. Changes in the transcript levels of many genes involved in sucrose catabolism and nitrogen assimilation and utilization were also observed between control and PEG-treated embryos.

Improving tolerance of somatic emrbyos of Picea glauca to flash desiccation with a cold treatment (desiccation after cold acclimation)

Controlled mild desiccation of mature white spruce somatic embryos prior to germination improves the quality of the germinated embryos. More severe desiccation results in increased injury and death but is desirable for long-term storage of embryos and production of desiccated artificial seed. A method was developed to improve desiccation tolerance in somatic embryos using a temperature treatment. Culture plates with embryos at four stages of development were subjected to temperatures of 1, 5, 10, or 20°C for periods of 0, 1, 2, 4, or 8 wk duration. After the temperature treatment, the embryos were harvested and air-dried for 2h under a laminar flow hood. Dried embryos were placed directly on germination medium and the quality of the germinants was assessed after 4 wk. The initial maturation stage of the embryo and the temperature and duration of the treatment had a significant effect on the quality of the germinants. Most treatments caused marked differential survival of organs. The optimal response was obtained with embryos that had been grown for 51 d (cotyledonary stage) on maturation medium and that were subsequently exposed to a temperature of 5°C for 8 wk prior to air drying. This treatment produced 58% undamaged germinants with normal cotyledons, hypocotyls, and roots. Only 1% of the untreated air-dried embryos germinated normally.

Ascorbic acid changes the pattern of purine metabolism during germination of white spruce somatic embryos

It has previously been shown that exogenous applications of ascorbic acid (AA) increase the conversion frequency of somatic embryos of white spruce (Picea glauca (Moench) Voss). To determine whether ascorbic acid alters purine metabolism during the early phases of embryo germination, the relative rates of purine salvage and degradation were investigated by following the metabolic fates of exogenously applied [8-14C]adenine, [8-14C]adenosine, and [8-14C]inosine, and the activities of several key enzymes. We demonstrated that both the salvage and the degradation pathways operate during germination. Specifically, adenine and adenosine were mainly salvaged to nucleotides and nucleic acids, whereas an appreciable amount of inosine was degraded to CO2 and ureides. Comparisons of purine metabolism between control and AA-treated embryos showed that exogenous applications of ascorbic acid enhanced the ability of the embryos to take up adenine and adenosine throughout the germination period. Furthermore, the higher enzymatic activities of adenosine kinase and adenine phosphoribosyltransferase were responsible for the larger proportion of adenine and adenosine being salvaged in AA-treated embryos compared with control embryos. Thus, there was a positive correlation between the ability to anabolize purine precursors and successful embryo conversion.

The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos.

The present study was conducted to understand the role of sucrose in the medium on the maturation of black spruce and white spruce somatic embryos. A maturation medium containing 6% sucrose, which hydrolyzed into glucose and fructose, gave significantly more embryos than a medium containing 3.16% of each glucose and fructose. Preventing the complete sucrose hydrolysis by a daily transfer of the tissues onto fresh medium significantly decreased the yield of somatic embryos compared to when sucrose was allowed to complete its hydrolysis. This reduction was not due to the manipulation of the tissues during the transfer, since a daily in situ transfer did not affect embryo production. To verify if the better embryo production observed on a medium containing 6% sucrose was due to the increasing osmotic pressure of the medium, this increasing osmotic pressure was simulated with a sequence of media containing different concentrations of glucose and fructose. Unexpectedly and for both species, this simulation did not improve somatic embryo production, which stayed similar to the one obtained on constant osmotic pressure. To understand these results, embryos produced on the different treatments were analyzed in terms of sucrose, glucose, fructose and starch levels and protein contents. The embryo carbohydrate content was independent from the carbohydrate used in the maturation medium. However, embryos matured on 6% sucrose allowed to hydrolyze during the maturation period contained significantly more soluble and insoluble proteins than embryos matured on any other treatment. Furthermore, embryos with a higher protein content also exhibited a higher epicotyl appearance frequency. The role of sucrose as a regulatory factor during the maturation of spruce somatic embryos is discussed.

Purine metabolism during white spruce somatic embryo development: salvage of adenine, adenosine, and inosine

Contribution of the adenine, adenosine and inosine salvage to the purine nucleotide and nucleic acid biosynthesis during white spruce ( Picea glauca) somatic embryo maturation was estimated by in situ assays using [8- 14C]adenine, [8- 14C]adenosine and [8- 14C]inosine. The salvage of adenine and adenosine was high during the initial stages of embryo maturation, characterized by rapid cell proliferation, but it declined upon further embryo development. Inosine salvage activity was always much lower than that observed for adenine and adenosine. Consistent with these results, activities of adenine phosphoribosyltransferase (APRT) and adenosine kinase (AK) measured in the embryo extracts in vitro were much higher than the activity of inosine kinase (IK) during all stages of embryo development. Utilization of adenosine and inosine for nucleotide and nucleic acid synthesis was found to be regulated by the enzymes AK and IK, as the pattern of their activities was very similar to the activity of adenosine and inosine salvage, estimated with exogenously supplied precursors. However, little correlation between salvage of adenine and activity of APRT was found throughout somatic embryo maturation. As no adenosine nucleosidase activity was found in white spruce embryos, adenosine, but not adenine, seems to be the major end product of adenylate catabolism and becomes the predominant substrate for purine salvage in vivo. Thus, adenosine salvage appeared to have the most important role in white spruce embryos. Studies on the metabolic fate of [8- 14C]adenine and [8- 14C]adenosine suggest that turnover of adenine nucleotides is rapid, as some of them are utilized for nucleic acid synthesis. In contrast, most of [8- 14C]inosine taken up by the embryos seems to be directly catabolized by the conventional purine catabolic pathway via ureides in all stages of embryo maturation.

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

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.

Ascorbic acid metabolism during white spruce somatic embryo maturation and germination

Changes in ascorbic acid (AA) metabolism were investigated in 3 different cell lines of white spruce; a non‐embryogenic line (NE)WS and two embryogenic lines with low (E)WS1 and high (E)WS2 percentage of embryo germination. During embryo maturation, a high AA/dehydroascorbate (DHA) ratio was observed in the two embryogenic cultures. The increment of this ratio was mainly ascribed to the high ability of the (E)WS1 and (E)WS2 lines to generate AA from its last precursor, l‐galactono‐γ‐lactone, since no increase in the activities of the AA‐recycling enzymes, i.e., AA free‐radical reductase (AFRR) and DHA reductase (DHAR), was observed as the embryos developed. During the partial drying treatment needed for successful embryo germination, the ability to de novo generate AA continued to increase in the embryos of the two embryogenic lines, although the total ascorbate pool (AA+DHA) sharply decreased. A reactivation of the overall AA metabolism was observed at the inception of germination, especially in the (E)WS2 embryos. These results suggest that (1) the ability to metabolically alter the AA/DHA ratio may be a fundamental requirement for somatic embryo maturation and (2) the restoration of the AA metabolism during the first days of germination may be necessary for successful embryo germination.

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

Summary Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce ( Picea glauca ). The contribution of the de 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- 14 C]orotic acid, an intermediate of the de novo pathway, and with [2- 14 C]uridine and [2- 14 C]uracil, substrates of the salvage pathways. The de novo pathway was very active throughout embryo development. More than 80 percnt; of [6- 14 C]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 14 CO 2 , 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 the de novo and salvage pathways of pyrimidine nucleotide biosynthesis in vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measured in 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.

Kinetics of ethylene biosynthesis and its effects during maturation of white spruce somatic embryos

The objective of the current investigation was to study the role of ethylene in the maturation of white spruce (Picea glauca [Moench.] Voss) somatic embryos. This was carried out by examining the effects of (1) 1-aminocyclopropane-1-carboxylic acid (ACC), a direct precursor of ethylene in plant tissue, (2) silver nitrate (AgNO 3 ), an inhibitor of ethylene action, (3) α-aminooxyamino acid (AOA), a potent inhibitor of ethylene biosynthesis, and (4) enrichment with ethylene. Ethylene biosynthesis was biphasic and gradually increased during embryo development, whereas endogenous ACC and N-malonylaminocyclopropane-1-carboxylic acid (mACC) decreased. Addition of ACC or AOA to the culture medium increased or decreased, respectively, ethylene biosynthesis by altering endogenous ACC levels during the culture period. In contrast to AOA and AgNO 3 , ACC and ethylene enrichment significantly decreased the production of mature somatic embryos and increased the browning of the cultures. However, the structure of the shoot apex in mature cotyledonary stage embryos formed under ethylene enrichment was similar to that in control systems. This shows that a reduction in ethylene is beneficial to maturation of white spruce somatic embryos. This is further substantiated by the finding that the inhibitory effects of AOA were partially reversed by the addition of ethylene. The possible effects of the interaction between ethylene and polyamines on somatic embryo development are also discussed.

Mineral nutrition in white spruce ( Picea glauca [Moench] Voss) seeds and somatic embryos. I. phosphorus, phytic acid, potassium, magnesium, calcium, iron and zinc

The mineral nutrient storage reserves in white spruce ( Picea glauca [Moench] Voss) somatic embryos and various parts of white spruce seeds were investigated. Somatic embryos are embryos produced in tissue culture. A single somatic embryo or zygotic embryo contained similar amounts of P and both types of embryos had similar amounts of the total P present in phytic acid (PA). The large female gametophyte tissue, which is absent in a somatic embryo, contained 86% of the total P and 95% of the total phytic acid-phosphorus (PA-P) in a single seed. The female gametophyte of a single seed contained ≈7 times more P and ≈22 times more PA-P than a single zygotic embryo. Somatic and zygotic embryos had significantly less of their P present in PA than did the female gametophyte tissue, which has over 87% of its P present in PA. A single somatic embryo or zygotic embryo contained similar amounts of K, Mg, Ca, Fe and Zn. The female gametophyte tissue contained 59% of the K, 85% of the Mg, 28% of the Ca, 69% of the Fe, and 65% of the Zn in a single seed. The seed coat of a single seed contained the majority of a seed’s Ca (71%) and also contained significant levels of K, Fe and Zn. On a dry weight (DW) basis, differences were found in the amounts of all the mineral nutrients studied here because somatic embryos are larger than zygotic embryos. Overall, a white spruce somatic embryo has equivalent amounts of all these nutrients as found in a natural white spruce zygotic embryo.