Metabolic footprinting study of white spruce somatic embryogenesis using NMR spectroscopy.

White spruce is an important commercial species for reforestation. The success in its propagation through somatic embryogenesis is well documented; however the physiological processes involved are poorly understood and remain unoptimized. The variable quality embryos generated in vitro from the same genotype suggest control at the protein and metabolite level. In order to probe metabolic changes, we have conducted a "metabolic footprinting" study, whereby culture media from growing cells was quantitatively analyzed to determine which metabolites were consumed and excreted. Such experiments are advantageous in that there is no need to quench cellular metabolism or extract intracellular metabolites through time-consuming protocols. In this paper we demonstrate the application of the footprinting assay to somatic embryo cells of white spruce (Picea glauca) using 1D (1)H NMR spectroscopy. We have surveyed embryogenesis metabolism in two types of media, maintenance (MN) and maturation (MT). MN medium does not result in shoot apical meristem (SAM) formation, while MT medium induces the necessary changes leading to fully developed somatic embryos. The two types of media were easily distinguished using metabolomics analysis, namely multivariate pattern recognition statistics (orthogonal partial least squares discriminatory analysis). From this analysis, we have identified numerous compounds involved with branched chain amino acid pathways such as valine and isoleucine. These results are explained on the basis of known metabolic pathways implicated in plant and animal developmental processes, and ultimately implicate altered CoA biosynthesis.

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.

History of plant tissue culture.

Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. It owes its origin to the ideas of the German scientist, Haberlandt, at the begining of the 20th century. The early studies led to root cultures, embryo cultures, and the first true callus/tissue cultures. The period between the 1940s and the 1960s was marked by the development of new techniques and the improvement of those that were already in use. It was the availability of these techniques that led to the application of tissue culture to five broad areas, namely, cell behavior (including cytology, nutrition, metabolism, morphogenesis, embryogenesis, and pathology), plant modification and improvement, pathogen-free plants and germplasm storage, clonal propagation, and product (mainly secondary metabolite) formation, starting in the mid-1960s. The 1990s saw continued expansion in the application of the in vitro technologies to an increasing number of plant species. Cell cultures have remained an important tool in the study of basic areas of plant biology and biochemistry and have assumed major significance in studies in molecular biology and agricultural biotechnology. The historical development of these in vitro technologies and their applications are the focus of this chapter.

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.

History of plant tissue culture.

Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. It owes its origin to the ideas of the German scientist, Haberlandt, at the beginning of the 20th century. The early studies led to root cultures, embryo cultures, and the first true callus/tissue cultures. The period between the 1940s and the 1960s was marked by the development of new techniques and the improvement of those already in use. It was the availability of these techniques that led to the application of tissue culture to five broad areas, namely, cell behavior (including cytology, nutrition, metabolism, morphogenesis, embryogenesis, and pathology), plant modification and improvement, pathogen-free plants and germplasm storage, clonal propagation, and product (mainly secondary metabolite) formation, starting in the mid-1960s. The 1990s saw continued expansion in the application of the in vitro technologies to an increasing number of plant species. Cell cultures have remained an important tool in the study of basic areas of plant biology and biochemistry and have assumed major significance in studies in molecular biology and agricultural biotechnology. The historical development of these in vitro technologies and their applications are the focus of this chapter.

Clonal propagation of softwoods.

Softwoods or gymnosperms, which make up 60% of the forested areas of the world, are economically important as a source of lumber, pulp, and paper. Reforestation is a major activity worldwide and the potential benefits of using clonal planting stock have long been recognized. Tissue culture clonal methods or micropropagation is a newer approach that can be achieved by enhancing axillary bud breaking, production of adventitious buds (organogenesis), and somatic embryogenesis. Plantlet production via organogenesis requires at least four stages: (1) bud induction on the explant, (2) shoot development and multiplication, (3) rooting of developed shoots, and (4) hardening of plantlets. Similarly, the production of plantlets via somatic embryogenesis, which has the potential to produce a larger number of plantlets, and in a shorter period of time, also requires several stages. These include (1) induction, maintenance, and proliferation of embryogenic tissue; (2) maturation (both morphological and physiological) of somatic embryos; and (3) germination and conversion of the somatic embryos. In this chapter, plantlet production via organogenesis from seedling and adolescent/mature explants and somatic embryogenesis from immature and mature seeds of white spruce (Picea glauca) are outlined.

Changes of purine and pyrimidine nucleotide biosynthesis during shoot initiation from epicotyl explants of white spruce (Picea glauca).

Nucleotide metabolism was investigated during white spruce organogenesis by following the metabolic fate of (14)C-labeled adenine, adenosine and inosine, as purine precursors, and orotic acid, uridine, and uracil, as pyrimidine intermediates. Key enzymes of purine and pyrimidine metabolism were also assayed during the organogenic process. White spruce epicotyl explants cultured on shoot-forming (SF) medium had a better ability to utilize adenine and adenosine for nucleotide and nucleic acid synthesis, compared to tissue cultured on non-shoot forming (NSF) medium. High levels of salvage products were observed in SF tissue after 10 days in culture, when shoot formation was initiated along the epicotyl axis of the explants. Such a differential utilization of purine precursors was mainly due to the higher specific activity of the two adenine and adenosine salvage enzymes, adenine phosphoribosyltransferase (APRT) and adenosine kinase (AK), measured in SF tissue. Similar catabolism of inosine was observed in both SF and NSF conditions during the 30 days of culture. For pyrimidines, the higher activities of the de novo, salvage, and degradation pathways observed in SF tissue, compared to NSF tissue throughout the course of the experiment, clearly denote a faster turnover of pyrimidine nucleotides in the former. Taken together, these results suggest that a better utilization of purine bases and nucleosides for nucleotide and nucleic acid synthesis, as well as a more rapid turnover of pyrimidine nucleotides, represent a physiological switch, which occurs during the initiation and continuation of the organogenic process in white spruce.

N-acetyl glutamate kinase from Daucus carota suspension cultures: embryogenic expression profile, purification and characterization.

In Daucus carota, N-acetylglutamate-5-phosphotransferase (NAGK; E.C. 2.7.2.8) specific activity was shown to correlate with the progression of somatic embryogenesis and was highest in the latter stages, where growth was most rapid. The enzyme was subsequently purified greater than 1200-fold using heat treatment, ammonium sulfate fractionation, gel filtration, anion exchange and dye ligand chromatography. Carrot NAGK was shown to have a subunit molecular weight of 31 kDa and form a hexamer. The Kms for NAG and ATP are 5.24 and 2.11 mM, respectively. Arginine (Arg) is a K-type allosteric inhibitor of the enzyme, and Hill coefficients in the order of 5 in the presence of Arg suggest that the enzyme is highly cooperative. D. carota NAGK does not bind to Arabidopsis thaliana PII affinity columns, nor does the A. thaliana PII increase NAGK specific activity, indicating its cellular location is probably different.

Nitrogen-15 NMR studies of nitrogen metabolism in Picea glauca buds.

In vivo (15)N nuclear magnetic resonance (NMR) as well as (15)N solid-state magic angle spinning (MAS) NMR spectroscopy were used to investigate nitrogen metabolism in cultured white spruce (Picea glauca) buds. Long-term as well as short-term experiments were carried out involving the use of inhibitors of the nitrogen pathways such as methionine sulfoximine (MSO), azaserine (AZA) and aminooxyacetate (AOA). Both in vivo and solid-state NMR showed that when MSO blocked glutamine synthetase (GS) no NH(4)(+) is incorporated. When glutamate synthase (GOGAT) is blocked by AZA there is some incorporation into glutamine (Gln), but very little into alpha-amino groups (glutamate, Glu). The transamination inhibitor AOA does not affect the metabolism of (15)NH(4)(+) into Gln and Glu, but blocks the production of arginine (Arg), as would be expected. Proline (Pro) and gamma-aminobutyric acid (GABA), which are produced directly from Glu without a transamination step, were not affected. The solid-state NMR experiments showed that protein synthesis occurred. Collectively, our results show that NH(4)(+) can only be assimilated through the GS/GOGAT pathway in P. glauca buds.

Alterations in pyrimidine nucleotide metabolism as an early signal during the execution of programmed cell death in tobacco BY-2 cells.

Changes in pyrimidine metabolism 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 pyrimidine nucleotides was estimated by following the metabolic fate of the (14)C-labelled orotic acid, whereas the rates of salvage and degradation pathways were studied by measuring the respective incorporation of (14)C-labelled uridine and uracil under different treatments. Nucleic acid metabolism was also examined using labelled thymidine as a marker. The results show that specific alterations in the balance of pyrimidine nucleotide synthesis, which include a decreased rate of salvage activity of uracil and uridine and increased salvage activity of thymidine, represent a metabolic switch that establishes proper cellular conditions for the induction of PCD. In particular, a reduction in the utilization of uracil for salvage products occurs very early during PCD, before the appearance of typical cytological features of the death programme, thus representing an early metabolic marker for PCD. These changes are strictly associated with PCD, since they do not occur if NO or hydrogen peroxide are increased individually, or if actinomycin, which inhibits the death programme, is added into the medium in the presence of NO and hydrogen peroxide. The possible roles of these fluctuations in pyrimidine metabolism on the cellular nucleotide pool are discussed in relation to the induction of cell death.

Purine and pyrimidine nucleotide synthesis and degradation during in vitro morphogenesis of white spruce (Picea glauca).

In the last few years, somatic embryogenesis and organogenesis of white spruce (Picea glauca) have been used as model systems to investigate biochemical and physiological events related to morphogenesis. This has been possible because studies conducted in vitro allow for manipulations of the culture conditions, in which changes in morphogenetic events can be easily related to physiological alterations. De-novo synthesis, salvage, and degradation of both purine and pyrimidine nucleotides are operative at all stages of somatic embryo maturation and germination. Fluctuations in the activity of these pathways delineate important morphogenic events. The early phases of embryo development are accompanied by a decreased salvage activity of purine nucleotides, which reflects a reduction of cell proliferation and the initiation of organized growth. Activities of the salvage enzymes are present throughout the maturation period, and also during the imposition of the drying period, which is required for successful embryo germination. The operative salvage pathway in dried embryos is needed for the enlargement of the nucleotide pool necessary to sustain the reactivation of the overall cellular metabolism at germination, before the reactivation of the de-novo pathway, which is a later event. Manipulations of the culture medium which improve the germination frequency of the embryos also result in increased salvage activity. Similar changes in nucleotide synthesis were also observed during the initiation of shoot development from epicotyl explants of white spruce and cotyledons of radiate pine. Results from these studies can be used for improving growth and development in culture.

Purine and pyrimidine nucleotide metabolism in higher plants.

Purine and pyrimidine nucleotides participate in many biochemical processes in plants. They are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products, such as sucrose, polysaccharides, phospholipids, as well as secondary products. Therefore, biosynthesis and metabolism of nucleotides are of fundamental importance in the growth and development of plants. Nucleotides are synthesized both from amino acids and other small molecules via de novo pathways, and from preformed nucleobases and nucleosides by salvage pathways. In this article the biosynthesis, interconversion and degradation of purine and pyrimidine nucleotides in higher plants are reviewed. This description is followed by an examination of physiological aspects of nucleotide metabolism in various areas of growth and organized development in plants, including embryo maturation and germination, in vitro organogenesis, storage organ development and sprouting, leaf senescence, and cultured plant cells. The effects of environmental factors on nucleotide metabolism are also described. This review ends with a brief discussion of molecular studies on nucleotide synthesis and metabolism.

Pyrimidine nucleotide and nucleic acid synthesis in embryos and megagametophytes of white spruce (Picea glauca) during germination.

Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophytes of white spruce by following the metabolic fate of 14C-labelled orotic acid, uridine, and uracil, as well as by measuring the activities of the major enzymes participating in nucleotide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autoradiographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential utilization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may represent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of 14C-thymidine for nucleic acid synthesis increased in both dissected embryos and megagametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reactivation was a later event, and the root meristem became activated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germination.

Root induction in Pinus ayacahuite by co-culture with Agrobacterium tumefaciens strains.

Transformation of in-vitro-derived shoots of Pinus ayacahuite Ehrenb. was achieved by co-culture with an oncogenic strain (A281 x 200) of Agrobacterium tumefaciens. During co-culture rooting also occurred; however, this rooting was not induced by genetic transformation of host cells, because a "disarmed" strain of A. tumefaciens (EHA101) also induced rooting. Furthermore, direct contact between shoots and bacterial cells was not required. Rooting occurred in agar-solidified medium and in a soilless substrate (9:1 vermiculite:peat mix). We conclude that A. tumefaciens strains induced rooting in P. ayacahuite through a change in the rhizosphere, probably by producing some root-inducing compound(s), and not through transformation of host cells.