Journal of Experimental Botany, Vol. 53, No. 370, pp. 891-904,
April 15, 2002
© 2002 Oxford University Press
Original Papers |
Molecular and enzymatic analysis of ammonium assimilation in woody plants
Departamento de Biología Molecular y Bioquímica, Instituto Andaluz de Biotecnología, Unidad Asociada UMA-CSIC, Universidad de Málaga, E29071-Málaga, Spain
Received 18 July 2001; Accepted 5 November 2001
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Abstract |
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 Top Abstract IntroductionCharacteristics of GS, GOGAT...Expression studies as an...Modification of nitrogen...Future prospectsReferences |
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Ammonium is assimilated into amino acids through the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) enzymes. This metabolic pathway is driven by energy, reducing power and requires the net supply of 2-oxoglutarate that can be provided by the reaction catalysed by isocitrate dehydrogenase (IDH). Most studies on the biochemistry and molecular biology of N-assimilating enzymes have been carried out on annual plant species and the available information on woody models is far more limited. This is in spite of their economic and ecological importance and the fact that nitrogen is a common limiting factor for tree growth. GS, GOGAT and IDH enzymes have been purified from several woody species and their kinetic and molecular properties determined. A number of cDNA clones have also been isolated and characterized. Although the enzymes are remarkably well conserved along the evolutionary scale, major differences have been found in their compartmentation within the cell between angiosperms and conifers, suggesting possible adaptations to specific functional roles. The analysis of the gene expression patterns in a variety of biological situations such as changes in N nutrition, development, biotic or abiotic stresses and senescence, suggest that cytosolic GS plays a central and pivotal role in ammonium assimilation and metabolism in woody plants. The modification of N assimilation efficiency has been recently approached in trees by overexpression of a cytosolic pine GS in poplar. The results obtained, suggest that an increase in cytosolic GS might lead to a global effect on the synthesis of nitrogenous compounds in the leaves, with enhanced vegetative growth of transgenic trees. All these data suggest that manipulation of cytosolic GS may have consequences for plant growth and biomass production.
Key words: Gymnosperms, nitrogen assimilation, nitrogen recycling, transgenic trees, woody angiosperms.
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Introduction |
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TopAbstractIntroductionCharacteristics of GS, GOGAT...Expression studies as an...Modification of nitrogen...Future prospectsReferences |
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Ammonium and nitrate ions are present in the soils of boreal forests although they are usually available in low abundance (Martin and Lorillou, 1997
). As a result, trees have developed mycorrizhal associations to increase the efficiency of nitrogen assimilation (Martin and Lorillou, 1997). In fact, N availability is a common limiting factor for tree growth and development. In conifer forests, low soil pH, high residual content of lignin and other secondary plant products in the soil limit nitrification. Consequently, ammonium is the predominant source of N for tree nutrition (Berg, 1986). It is well documented that conifers, unlike herbaceous plants, have a preference for ammonium over nitrate as the N source (Bedell et al., 1999). Plant metabolic activities also release ammonium through processes such as protein/nucleic acid breakdown and subsequent amino acid/nucleotide catabolism, photorespiration and, the biosynthesis of phenylpropanoids. A product of the phenylpropanoid pathway is lignin, the second most abundant organic compound in the biosphere after cellulose. Lignin constitutes a major fraction in wood and therefore ammonium released in lignin biosynthesis is of quantitative importance during the life of a tree. Primary nitrogen assimilation from the soil and reassimilation into biomolecules are crucial processes for plant N economy, however, in many woody plants N assimilated from soil may not be utilized immediately, but can be stored for use during the next growing season (Coleman et al., 1994). Thus, for woody plants, the study of ammonium metabolism is of particular significance for the understanding of fundamental tree biology.
The incorporation of ammonium into the pool of N-containing molecules is first catalysed by the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle (Fig. 1). In this metabolic pathway GS (EC 6.3.1.2) catalyses the amidation of glutamate to generate glutamine at the expense of ATP hydrolysis. The second enzyme, GOGAT (EC 1.4.7.1; 1.4.1.14), is responsible for the reductive transfer of amide N to 2-oxoglutarate for the generation of two molecules of glutamate, one of which is recycled for glutamine biosynthesis (Miflin and Lea, 1980). This N assimilatory pathway is driven by energy and reducing power derived from photosynthesis or from the catabolism of protein and carbon reserves. Glutamate and glutamine are the N donors for the biosynthesis of major N compounds in plants including other amino acids, nucleic acids bases, polyamines, and chlorophylls. For instance, N can be channelled to the biosynthesis of aspartate and asparagine catalysed by aspartate amino transferase (AspAT) (EC 2.6.1.1) and asparagine synthetase (AS) (EC 6.3.5.4), respectively (Fig. 1). Recent molecular studies using defective mutants supported the in vivo roles of AspAT in N metabolism (Schultz et al., 1998). In most plants the amides glutamine or asparagine are important vehicles for N transport between source and sink tissues.
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The stoichiometry of the GS/GOGAT cycle clearly shows that the incorporation of ammonium for the net synthesis of glutamate requires the supply of 2-oxoglutarate (Fig. 1
). This oxoacid is provided by the reaction catalysed by NAD+- or NADP+-dependent isocitrate dehydrogenase (IDH) (EC 1.1.1.41; 1.1.1.42).
In addition to GS, GOGAT and IDH, another enzyme potentially involved in ammonium metabolism is NADH-glutamate dehydrogenase (GDH) (EC 1.4.1.2). GDH can catalyse the reductive amination of 2-oxoglutarate and the reverse catabolic reaction of oxidative deamination of glutamate. GDH is located in the mitochondrial matrix where it is mainly responsible for glutamate catabolism under carbon and N-limiting conditions (Stewart et al., 1995; Aubert et al., 2001). The enzyme may also function in the direction of glutamate biosynthesis when ammonium is highly abundant (Melo-Oliveira et al., 1996). The controversial role of GDH in the assimilation of ammonium has been discussed (Miflin and Habash, 2002).
The biochemistry and molecular biology of N-assimilating enzymes in plants has been extensively studied and recent comprehensive reviews are available (Lam et al., 1996; Temple et al., 1998; Ireland and Lea, 1999; Gálvez et al., 1999). However, most studies have focused on annual plants, thus available information concerning nitrogen assimilation in woody models is much more limited, particularly at the molecular level (Cánovas et al., 1998). This is in spite of the economic and ecological importance of these plants. In this paper the current status of research on ammonium assimilation and metabolism in woody plants is reviewed. In the first section the molecular characterization of the pathway in different woody plants is presented as well as subcellular localization in angiosperms and gymnosperms. In the second section, gene expression analysis and distribution in different cellular types is discussed with regard to the functional roles of the N-assimilating enzymes. Initial studies on genetic manipulation addressed to increase growth rate in trees are also presented. The final section includes future prospects for N assimilation studies in woody plants and potential applications.
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Characteristics of GS, GOGAT and IDH |
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TopAbstractIntroductionCharacteristics of GS, GOGAT...Expression studies as an...Modification of nitrogen...Future prospectsReferences |
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Annual plants
The metabolic requirement for GS activity in ammonium assimilation in plants is fulfilled by GS isoforms expressed in specific organs and at specific developmental stages (see Cren and Hirel, 1999
; Ireland and Lea, 1999, for recent reviews). With regard to the subcellular localization, two different classes of GS have been reported in angiosperms: GS1 in the cytosol and GS2 in the chloroplasts. In the leaves of many plants GS2 is the predominant isoform where it is located in mesophyll photosynthetic cells, whereas GS1 is generally less abundant in photosynthetic tissues. The exceptions include C4 plants, which exhibit low photorespiratory activity, and C3 plants under certain physiological conditions, such as senescence and during response to biotic or abiotic stresses (Cren and Hirel, 1999). GS1 is the predominant isoform in roots and other non-photosynthetic tissues (Ireland and Lea, 1999), where it is located in cells of the vascular bundles (Edwards et al., 1990; Dubois et al., 1996). However, GS2 has also been identified in roots of legume plants grown in the presence of nitrate (Woodall and Forde, 1996), which could represent a physiological adaptation to nitrate-rich soils. In addition, nodule-specific GS1 is involved in post-N-fixation ammonium assimilation (Ireland and Lea, 1999). The complete amino acid sequence of cytosolic and plastidic GS subunits has been deduced from the corresponding cDNAs isolated from many annual plant species including major crops (Cren and Hirel, 1999; Ireland and Lea, 1999). In most plants examined, chloroplastic GS (GS2) is encoded by a single nuclear gene and its functional role is the assimilation of ammonium derived from the reduction of nitrate and from photorespiration (Lam et al., 1996). Cytosolic GS (GS1) exists as a variable number of isoforms which are encoded by a small gene family whose members are differentially expressed during development, or in response to external stimuli (Ireland and Lea, 1999). Unlike GS2, the physiological roles of individual genes encoding cytosolic isoforms (GS1) are unclear, but recent reports indicate that GS1 may be involved in a variety of processes, including the primary assimilation of ammonium from the soil (Sakakibara et al., 1996), N recycling and translocation between source and sink tissues in the plant, and reassimilation of N mobilized during senescence (Cren and Hirel, 1999).
In plants, glutamate synthase (GOGAT) occurs as two distinct molecular forms which differ with respect to the source of reductant for enzyme catalysis: NADH-GOGAT and ferredoxin (Fd)-GOGAT. Both enzymes display different physico-chemical, immunological and regulatory properties and are encoded by separate genes (Temple et al., 1998; Ireland and Lea, 1999). Fd-GOGAT is an iron–sulphur flavoprotein, plastid-located and represents the predominant molecular form in photosynthetic tissues although its presence has also been reported in roots and nodules (Lam et al., 1996; Temple et al., 1998). In most plants analysed, Fd-GOGAT is encoded by a single gene, however, in Arabidopsis two genes have been characterized (Coschigano et al., 1998): GLU1 is exclusively expressed in the leaf and is light-regulated, whereas GLU2 is expressed in leaves and roots and is not regulated by light. The expression pattern of the genes and the physiological characterization of defective mutants support a role of GS2 and Fd-GOGAT in the assimilation of ammonium derived from the reduction of nitrate and from photorespiration (Coschigano et al., 1998; Ireland and Lea, 1999). NADH-GOGAT, also an iron–suphur flavoprotein, is present at low abundance in leaves, but it is more abundant in non-photosynthetic tissues such as roots and nodules, where it is located in non-chlorophyllous plastids (Temple et al., 1998). The structure of the alfalfa gene encoding NADH-GOGAT has been reported (Temple et al., 1998) and its expression is restricted to root nodules where it plays a significant role in the assimilation of ammonium derived from symbiotic N2 fixation (Trepp et al., 1999). The localization of GS1 and NADH-GOGAT proteins in the root vascular bundles of rice supports the possibility of a co-ordinated function in the assimilation of ammonium in roots (Ishiyama et al., 1998).
Plant IDHs differ in the pyridine nucleotide they use as co-substrate and also in their localization in the cell. The mitochondrial NAD+-dependent IDH is the enzyme involved in the Krebs cycle, while NADP+-dependent IDH exists in different subcellular compartments, including the cytosol, chloroplasts, peroxysomes, and mitochondria. Cytosolic NADP+-IDH is the most active IDH enzyme in both angiosperms and gymnosperms, and it has been suggested to be the main enzyme involved in providing carbon skeletons for N assimilation when large amounts of 2-oxoglutarate are required (Chen and Gadal, 1990). Therefore, the supply of 2-oxoglutarate through a cytosolic pathway involving aconitase and NADP+-IDH represents an alternative route to the Krebs cycle enzymes, for providing carbon skeletons for ammonium assimilation and the biosynthesis of glutamate and glutamine (Chen and Gadal, 1990; Gálvez et al., 1999) (Fig. 2). However, and regardless of its origin, mitochondrial or cytosolic, 2-oxoglutarate must be transported into the chloroplast for glutamate biosynthesis. Thus, it is possible that transport of metabolites across the membranes could be a limiting step in ammonium assimilation rather than 2-oxoglutarate biosynthesis (Gálvez et al., 1999). The biosynthesis of aspartate and asparagine is also dependent of carbon provision in the form of oxalacetate (Fig. 2). Therefore, the supply of carbon skeletons for amino acid biosynthesis requires the flow of carbon metabolites into the Krebs cycle to avoid depletion of intermediates. This requirement must be met by increasing carbon flux via glycolysis and phosphoenolpyruvate carboxylation. The metabolism of these intermediary compounds tightly links nitrogen assimilation and carbon metabolism.
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Woody angiosperms
GS has been characterized from angiosperm woody species including apple (Malus domestica) (Titus and Kang, 1982), avocado (Persea americana) (Loulakakis et al., 1994), grapevine (Vitis vinifera) (Loulakakis and Roubelakis-Angelakis, 1996), rubber tree (Hevea brasilensis) (Pujade-Renaud et al., 1997), black walnut (Juglans nigra) (Simonson and Twigg, 1999), hybrid poplar (Populus tremulaxP. alba) (Gallardo et al., 1999), and the root nodules of alder (Alnus glutinosa) in symbiosis with Frankia (Hirel et al., 1982; Guan et al., 1996) (Table 1). Available biochemical data and the molecular characterization of GS cDNA clones indicate the existence of chloroplastic and cytosolic isoenzymes possibly encoded by a small gene family as reported for herbaceous angiosperms.
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Two complementary DNA (cDNA) clones encoding Fd-dependent glutamate synthase have been characterized from grapevine and the deduced amino acid sequence of the enzyme is significantly similar to the respective sequences of other plant Fd-GOGATs (Loulakakis and Roubelakis-Angelakis, 1997
). The existence of two Fd-GOGAT genes in the grapevine genome was inferred by Southern blot analysis using the isolated cDNAs as molecular probes (Loulakakis and Roubelakis-Angelakis, 1997). It is unknown whether or not they are equivalent to the two Fd-GOGAT genes in Arabidopsis. NADH-GOGAT activity was extracted from the bark tissue of the apple, partially purified and its kinetic parameters determined although the enzyme has not been characterized at the molecular level (Titus and Kang, 1982).
Recently, a cDNA clone encoding NADP+-dependent IDH from an eucalypt (Eucalyptus globulus) has been reported (Boiffin et al., 1998). The deduced protein lacks an amino terminal transit peptide and shows the highest similarity to plant cytosolic IDH. The enzyme was preferentially localized in the epidermis and vascular elements of the root, where its relative abundance was enhanced by ectomycorrhizal colonization. These findings suggest a role of the enzyme in providing carbon skeletons for the assimilation of N translocated from the fungal partner.
Gymnosperms
The enzymes characterized in gymnosperms are summarized in Table 1. GS has been purified from needles and roots of jack pine (Pinus banksiana) and from roots of Douglas fir (Pseudotsuga menziesii), and its physico-chemical and kinetic properties have been determined (Vézina and Margolis, 1990; Bedell et al., 1995). With regard to the subcelullar localization of the enzyme, in the photosynthetic tissues of pine seedlings and other conifers, only cytosolic isoforms of GS (GS1) have been identified (Cánovas et al., 1991; Cantón et al., 1993, 1996; Avila et al., 1998). The chloroplastic isoform (GS2) has not yet been detected by using a number of different molecular approaches including separation of isoforms by ion-exchange chromatography, Western blot analysis, and screening and random sequencing of clones in pine cDNA libraries. The localization of the GS protein exclusively in the cytosol of photosynthetic and non-photosynthetic pine cells was demonstrated by immunocytolocalization (García-Gutiérrez et al., 1998). These data indicate that glutamine biosynthesis occurs in the cytosol of pine cells, not only during the initial stages of pine development, but also in pine trees (Avila et al., 2000). However, the presence of GS2 has been reported in the leaves of ginkgo (Ginkgo biloba), a non-coniferous gymnosperm (García-Gutiérrez et al., 1998).
In cotyledons of Scots pine (Pinus sylvestris) two GS isoforms, GS1a and GS1b, have been reported (Cantón et al., 1993; Avila et al., 1998) which exhibit differential chromatographic behaviours and are composed of subunits of a similar size, but different charge. GS1a is predominant in pine cotyledons (Cantón et al., 1993), while GS1b is a minor form whose relative amount increases following phosphinothricin (PPT) treatment (Avila et al., 1998). PPT is a structural analogue of glutamate that behaves as a powerful and irreversible inhibitor of GS activity. Full-length cDNA clones encoding these two cytosolic isoforms have been isolated and the deduced amino acid sequences analysed (Cantón et al., 1993; Elmlinger et al., 1994; Avila et al., 2000). GS1a and GS1b polypeptides lack N-terminal presequences confirming their assembly into cytosolic oligomeric enzymes. This inference is supported by comparative analysis with the GS amino acid sequences from angiosperms. Interestingly, GS1a contains amino acid residues characteristic of only the GS2 polypeptide, including cysteine residues usually absent in the cytosolic polypeptides. By using site directed mutagenesis it was determined that Cys249 is involved in enzyme stability (A García-Gutiérrez, FM Cánovas, unpublished results). GS1a and GS1b genes are closely linked in the genome, supporting a proposed origin of GS isogenes by adjacent gene duplication. GS1b is more similar to cytosolic GS from angiosperms than to GS1a. The study of phylogenetic relationships between plant GS genes suggest the possibility that angiosperms might have received an ancestral GS1 gene more closely related to the Pinus GS1b, whereas GS1a is unique to gymnosperms (Avila et al., 2000). An extension of this study, including analysis of GS sequences from plants, algae and prokaryotes, showed that GS2 possibly evolved from a duplicated GS1 gene long before the gymnosperms/angiosperms divergence (Fig. 3). Although expression of a GS2 gene has not been detected in conifers, these findings are consistent with the presence of a GS2 enzyme in the gymnosperm G. biloba (García-Gutiérrez et al., 1998).
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Fd-GOGAT has also been characterized in gymnosperms. As found in angiosperms, Fd-GOGAT is a single polypeptide of about 168 kDa in pine (P. pinaster and P. sylvestris), pinsapo fir (Abies pinsapo), larch (Larix decidua) and ginkgo (García-Gutiérrez et al., 1995
, 1998). Several cDNA clones encoding the C-terminal region of pine Fd-GOGAT were isolated from an expression library from P. sylvestris seedlings by screening with affinity-purified antibodies against the enzyme (García-Gutiérrez et al., 1995). The pine polypeptide contains three conserved cysteine residues involved in an iron–sulphur cluster and a flavin mononucleotide binding site consisting of two putative domains conserved in other plant Fd-GOGATs (Temple et al., 1998). Comparison of the pine amino acid sequence with monocot and dicot sequences available in the data bank, revealed that the primary structure of Fd-GOGAT is remarkably well conserved in angiosperms and gymnosperms, in spite of the evolutionary distance (García-Gutiérrez et al., 1995). Results derived from Southern blot analysis are consistent with the existence of more than one gene for Fd-GOGAT in the pine genome (García-Gutiérrez et al., 1996). NADH-GOGAT activity has also been detected in young pine seedlings (Elmlinger and Mohr, 1991; García-Gutiérrez et al., 1995), but no molecular studies of the enzyme have yet been performed.
NADP+-IDH has been purified and characterized from Scots pine (P. sylvestris). Only one form of cytosolic localization was detected in green cotyledons with molecular and kinetic properties similar to those described for NADP+-IDHs in angiosperms (Palomo et al., 1998). These data suggest that the enzyme is well conserved in plants and could play similar physiological roles in angiosperms and gymnosperms. Expression studies in different pine tissues during early development, suggest that in addition to providing 2-oxoglutarate for glutamate biosynthesis, NADP+-IDH may have other, as yet unknown, biological roles. NAD+-IDH and NADP+-IDH activities have also been studied in mitochondria purified from Norway spruce (Picea abies) seedlings (Cornu et al., 1996). Both enzymes were detected in the mitochondrial matrix fraction, but at different abundances, NAD+-IDH activity was about 2-fold more abundant than NADP+-IDH. Furthermore, kinetic differences in substrate affinities were observed. No further characterization at the molecular level has yet been undertaken.
The characterization of GS, GOGAT and IDH in angiosperm woody plants indicates that these enzymes may play similar physiological roles to those found in annual herbaceous plants. Thus in photosynthetic tissues, glutamine and glutamate synthesis is catalysed by the GS2/Fd-GOGAT cycle located within the chloroplast (Fig. 4A), although 2-oxoglutarate should be provided by cytosolic or mitochondrial IDHs. In the seedlings of the gymnosperm g. biloba, the subcellular localization of GS and GOGAT enzymes is similar to that found in angiosperms (Fig. 4A). However, in conifers, glutamine biosynthesis occurs in the cytosol and Fd-dependent glutamate synthase is a soluble enzyme located in the chloroplast stroma (García-Gutiérrez et al., 1995) (Fig. 4B). The separation of glutamine and glutamate biosynthesis in different subcellular compartments implies not only the compartmentation of the GS/GOGAT cycle but also implies that glutamine must be transported from the cytosol into the plastid for glutamate production. Recent studies indicate the existence of a translocator in the chloroplast membranes of P. pinaster, that may be responsible for the import of glutamine into the organelle in antiport with glutamate (MG Claros, FM Cánovas, unpublished data). It has been hypothesized that the distribution of glutamine biosynthesis in different cellular compartments may be associated with etiolation in seedlings (García-Gutiérrez et al., 1998). Chloroplast development in conifers is far less regulated by light than in ginkgo or in angiosperms. Conifers would present an ancestral pathway of seedling development in plants, while etiolation appeared later in evolutionary linneages (García-Gutiérrez et al., 1998). According to this hypothesis, glutamine and glutamate biosynthesis would be confined to the chloroplast of mesophyll cells in species with light-dependent chloroplast development, whereas compartmentation between cytosol and chloroplast could be required in species, with light-independent plastid development.
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Expression studies as an approach to identify functional roles of individual enzymes |
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TopAbstractIntroductionCharacteristics of GS, GOGAT...Expression studies as an...Modification of nitrogen...Future prospectsReferences |
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Effect of nitrogen nutrition and light
Expression analysis in grapevine revealed that two GS1 isogenes are highly expressed in roots and to a lower extent in shoots and leaves (Loulakakis and Roubelakis-Angelakis, 1996
). Both genes are differentially affected by exogenously supplied ammonium, whereas nitrate had no effect (Loulakakis and Roubelakis-Angelakis, 1996). However nitrate stimulated Fd-GOGAT expression (Loulakakis and Roubelakis-Angelakis, 1997). These data are in agreement with previous reports in annual herbaceous plants, indicating a role of specific members of the GS1 gene family in the assimilation of externally supplied ammonium in co-ordination with NADH-GOGAT (Ireland and Lea, 1999). The increase of Fd-GOGAT and GS2 expression in maize roots in response to exogenously supplied nitrate (Redinbaugh and Campbell, 1993) suggest a role of the plastid-located GS/GOGAT cycle in the assimilation of ammonium derived from nitrate reduction. By contrast to that found in angiosperm woody plants, N nutrition either as nitrate or ammonium has a limited role in the regulation of GS and Fd-GOGAT in developing seedlings of maritime (Cánovas et al., 1991; García-Gutiérrez et al., 1995) and Scots pine (Elmlinger and Mohr, 1991, 1992).
Light is another external stimulus regulating N-assimilating enzymes in plants. Thus, the expression of genes for GS2 and Fd-GOGAT is light-regulated, whereas for the GS1 isoenzymes and NADH-GOGAT little effect of illumination, if any, has been described (Lam et al., 1996). Light enhancement of grapevine Fd-GOGAT expression was observed at the mRNA and enzyme activity level, but the transcript was also present at lower abundance in dark-grown plants (Loulakakis and Roubelakis-Angelakis, 1997). Light strongly stimulated GS1a mRNA accumulation during the development of Scots pine cotyledons (Cantón et al., 1999). GS1a transcripts increased in dark-grown seedlings transferred to light and decreased in dark-adapted seedlings in a similar way to mRNAs for photosynthesis genes such as rbcS and lhcb2. Functional expression analysis of the GS1a promoter in transgenic Arabidopsis, indicates it contains regulatory sequences involved in the response to light (Avila et al., 2001b). Regulation of conifer GS by light appears to be exerted by coaction of the phytochrome and cryptochrome photoreceptors (Elmlinger et al., 1994). In P. sylvestris seedlings, light is a major factor controlling the accumulation of Fd-GOGAT (Elmlinger and Mohr, 1991). However, P. pinaster seedlings accumulated Fd-GOGAT activity, polypeptide and transcript in a light-independent manner (García-Gutiérrez et al., 1995), suggesting a differential requirement of light among pine species for Fd-GOGAT accumulation during early seedling development.
Cellular distribution and developmental regulation
Expression of N-assimilating enzymes has been examined in detail during conifer seed germination and initial stages of seedling development. In loblolly pine (Pinus taeda), the breakdown of storage proteins during germination coincides with the accumulation of free amino acids in the seedling, particularly arginine, which is the predominant vehicle for N transport from the megagametophyte to the embryo (King and Gifford, 1997). Protein breakdown is accompanied by a marked increase in arginase activity. Urease activity has also been reported in pine seedlings confirming that arginine catabolism is an important source of ammonium during the early growth of pine seedlings (Todd et al., 2001). The roles of the two GS1 genes (GS1a and GS1b) in N flow from the seed to the developing seedling have been recently reported for Scots pine (Avila et al., 2001a). GS1b is the functional gene at the early stages of germination providing the organic N necessary for de novo protein biosynthesis and is possibly related to the loss of seed dormancy (Schneider and Gifford, 1994). High levels of GS1b expression in the medullar region of the hypocotyl precedes formation of the first vascular elements suggesting that GS1b functions in N translocation in developing seedlings. By contrast, GS1a, Fd-GOGAT (García-Gutiérrez et al., 1995) and NADH-GOGAT (A García-Gutiérrez, FM Cánovas, unpublished data) expression is very low in the embryo and presumably the corresponding genes are not involved in glutamate biosynthesis in the embryo.
In seedlings, expression of GS1a is restricted to tissues containing chloroplasts, including cotyledons and the upper part of the hypocotyl (Cantón et al., 1993). Furthermore, in Scots pine GS gene expression in these tissues is strongly stimulated by light (Elmlinger et al., 1994; Cantón et al., 1999). These data support a role for GS1a in the generation of amino donors for the biosynthesis of major N compounds in photosynthetic tissues: a role similar to the physiological role of chloroplastic GS2 in angiosperms. By contrast, GS1b is highly abundant in hypocotyls and roots, although it is also present at low levels in the cotyledons. In all these tissues GS1b is associated with the vascular bundles (Avila et al., 2001a). This expression pattern is quite similar to that found for GS1 in angiosperms (Edwards et al., 1990; Dubois et al., 1996) and suggests that GS1b plays an important role in N transport and translocation within the seedling. Moreover, the presence of GS1b in the xylem of pine trees (Avila et al., 2000; FM Cánovas, C Avila, FR Cantón, unpublished results), suggests a functional role of this GS isoform in the reassimilation of ammonium released from lignin biosynthesis during wood formation. In green cotyledons, where GS1a and GS1b coexist, they show distinct distribution patterns, further supporting differentiated functions in N metabolism (Fig. 5). GS1a expression in cotyledons is well correlated with the reported abundance of arginase (Todd et al., 2001), suggesting that GS1a plays a primary role in the reassimilation of ammonium released in arginine metabolism. GS1a may also function in reassimilation of ammonium released in photorespiration in photosynthetic cells of pine seedlings; a metabolic role that chloroplastic GS (GS2) assumes in angiosperms. In this context, it is worth noting that arginine metabolism has been proposed to be involved in the N photorespiratory cycle (Ludwig, 1993). High levels of expression of Fd-GOGAT and NADP-dependent IDH (García-Gutiérrez et al., 1995; Palomo et al., 1998) suggest that a functional GS1a-GOGAT cycle is operative in green tissues of pine.
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Based on the abundance of asparagine in the later stages of seedling growth, it has been suggested that asparagine is the form of N that is transported from cotyledons to other parts of the pine seedling (King and Gifford, 1997
). Asparagine biosynthesis in plants is a glutamine-dependent reaction catalysed by the enzyme asparagine synthetase (Sieciechowicz et al., 1988). Thus, the generation of glutamine for asparagine biosynthesis could be undertaken by GS1b. The localization of GS1b in vascular cells, the same place where asparagine synthetase expression has been reported (Nakano et al., 2000), is consistent with this hypothesis. Moreover, the abundance of GS1b in the hypocotyl and roots and the precise localization of GS1b transcripts in the central cylinder of the root, further support such a role for GS1b in N transport to sink tissues. Recently, using an antisense approach, Brugière et al. reported an essential role of GS1 located in phloem cells in the production of proline (Brugière et al., 1999). Nevertheless, proline abundance is very low in germinating pines (King and Gifford, 1997), therefore, GS1b does not appear to be involved in the biosynthesis of this amino acid following storage protein breakdown. Biochemical and molecular data suggest the existence of two glutamine cycles in developing pine seedlings (Fig. 5): (1) a GS1a/GOGAT pathway implicated in ammonium assimilation and glutamate biosynthesis in mesophyll cells, where a high demand of N compounds would be required for cell growth and proliferation; and (2) a cytosolic GS1b/AS pathway involved in the biosynthesis of amides in vascular tissues for N transport to the growing apices.
Changes in gene expression in response to stress or senescence
The expression of GS1a in Scots pine appears to be dependent on factors associated with the integrity of developing chloroplasts (Cantón et al., 1999). In the presence of norfluorazon, a herbicide causing chloroplast damage by photo-oxidation as a result of carotenoid biosynthesis inhibition, light-grown plants showed a large decrease in the levels of GS1a, rbcS and lhcb2 mRNAs, whereas the abundance of the mitochondrial ß-ATP synthase was less affected. In tomato plants, infection by Pseudomonas syringae or treatment with the herbicide, phosphinothricin, a specific GS inhibitor, leads to chloroplast degeneration and apparent leaf chlorosis (Pérez-García et al., 1995, 1998a). When bacterial infection or herbicide treatment was carried out in the light, down-regulation of GS2 expression was observed and cytosolic GS1 appeared as the predominant GS polypeptide. These GS isoform replacements only occurred in illuminated leaves and were not observed during bacterial infection or during PPT treatment when photosynthetic activity was suppressed (Pérez-García et al., 1998a). This indicates that light-dependent factors are implicated in the regulation of expression of GS isoforms. The transient application of PPT to developing pine seedlings triggers the accumulation of the GS1b holoenzyme and, after separation by 2D-electrophoresis, specific induction of the GS1b polypeptide was observed (Avila et al., 1998). This response appears to be transcriptionally regulated (Avila et al., 2000) and is restricted to green tissues, suggesting a dependence on photosynthetic metabolism as reported for tomato (Pérez-García et al., 1998a). These data suggest that treatment with PPT provokes the loss of photosynthetic functions and subsequent chloroplast degeneration.
To study further the effect of the herbicide in conifers, the precise localization of GS1b transcripts was determined in sections of pine cotyledons using in situ localization. As shown in Fig. 6, general enhancement of expression was found in all cellular types, but the induction of GS1b expression was mainly localized in cells of the photosynthetic parenchyma.
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These data are in agreement to the findings reported for annual plants. Pérez-García et al. demonstrated that the induced GS1 in response to PPT or bacterial infection was localized in mesophyll cells of tomato plants where it could play a role in the assimilation of N mobilized from chloroplast degradation (Pérez-García et al., 1998
b). Similar results were found in senescing tobacco leaves, where a parallel induction of GS protein expression in the mesophyll cell cytosol was observed with the progression of leaf senescence (Brugière et al., 2000). In tomato leaves, the newly synthesized GS1 polypeptide was shown to be a novel GS isoform different in charge from the constitutive GS present in untreated tissue (Pérez-García et al., 1998b). In tobacco, the new GS1 protein was identified as the product of the Gln1-3 gene, that is expressed in the cytosol of roots and flower cells (Dubois et al., 1996).
In addition to the central role of GS1 in N remobilization recent reports suggest the implication of other auxiliary enzymes in this process. For example, during natural senescence in tobacco, a strong correlation between proteolytic activity and both GS1 and GDH expression was observed (Masclaux et al., 2000). GDH may provide the required glutamate for glutamine biosynthesis when Fd-GOGAT activity is not present. In tomato plants infected with Pseudomonas, the induction of asparagine synthetase (AS) has been observed in parallel with the induction of GS1 expression. This suggests the existence of a functional GS1/AS cycle (F Olea, A Pérez-García, A de Vicente, FM Cánovas, unpublished data), as proposed for pine seedlings (Avila et al., 2001a; this work). These findings further suggest that the metabolic status of the leaf, in particular the C/N ratio, could be involved in the control of expression of genes involved in ammonium assimilation. This co-ordinated GS1/AS pathway can be operative for the assimilation of ammonium under specific conditions, such as natural or induced senescence, carbon starvation (Chevalier et al., 1996), response to water stress (Bauer et al., 1997), or N fixation (Trepp et al., 1999). The existence of an excess of N and/or a limitation of carbon is common to all these conditions.
In many trees the N present in leaves is mobilized during autumn and stored in perennial tissues to be remobilized at the beginning of the next growing season. Increased levels of N-assimilating enzymes such as GS and possibly GDH have been reported in apple (Titus and Kang, 1982), during the autumnal senescence of leaves before abscission. Although analysis of specific GS isoenzymes was not undertaken in this study, it is possible that cytosolic GS could be involved. Since this process is of great importance for overall N economy in trees, studies of N cycling and remobilization need to be re-examined by determining seasonal variations of gene expression for certain key enzymes. The activities and isoenzyme profiles of GS and GDH have been studied during development and ripening of avocado fruit (Loulakakis et al., 1994). No changes were apparent during fruit development. By contrast, steady-state levels of both enzymes were considerably altered during ripening. GDH expression increased during the ripening, whereas total GS activity declined (Loulakakis et al., 1994).
Hormonal regulation of ammonium assimilation in trees has been recently reported. In the latex of rubber tree, transcripts for the cytosolic GS accumulate in response to ethylene (Pujade-Renaud et al., 1997). Although initially the effect was observed in trees regularly submitted to tapping (wounding) (Pujade-Renaud et al., 1994), direct regulation by ethylene, independent of wounding, was also demonstrated. The observed induction required 6–12 h, a period possibly needed for transduction of the ethylene signal (Pujade-Renaud et al., 1997).
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Modification of nitrogen metabolism in transgenic woody plants |
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TopAbstractIntroductionCharacteristics of GS, GOGAT...Expression studies as an...Modification of nitrogen...Future prospectsReferences |
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A current focus in plant improvement is the increase in plant growth and biomass accumulation as a result of the modification of the expression of growth-related genes. In early studies of expression of chimeric genes encoding key N metabolism enzymes such as nitrate and nitrite reductase, GS and AS, no effect on the phenotype of the modified plants was observed (Foyer and Ferrario, 1994
). Co-transformation with other genes should be considered in order to avoid the possible generation of new limiting steps in the metabolic pathway. Nevertheless, more recent studies have shown that characteristics of agronomic importance can be introduced in transgenic herbaceous plants by the expression of heterologous GS isoenzymes. Thus, a higher capacity for photorespiration (Kozaki and Takeba, 1996), stimulation of seedling growth (Migge et al., 2000), and increase in tolerance to salt stress (Hoshida et al., 2000) have been reported using engineered plants which overexpress chloroplastic GS2. Furthermore, an increase in biomass production and acceleration of development have been observed in leguminous plants which overexpress cytosolic GS1 (Vincent et al., 1997; Limami et al., 1999).
Poplar is considered a model in molecular investigations of forest trees because of its small genome size, easy vegetative propagation and in vitro culture, and its amenability to transformation via Agrobacterium tumefaciens (Klopfenstein et al., 1997). The existence of a number of fast-growing hybrid poplar clones also permits short-term field trials to obtain results in a relatively short period of time in comparison with other trees (Klopfenstein et al., 1997).
The modification of N assimilation efficiency has recently been approached in trees by the overexpression of pine GS1a in hybrid poplar (Gallardo et al., 1999). An advantage in using woody plants in studying enhanced nitrogen assimilation is that the effect of the transgene expression can be accumulated in the tissues/organs for a long period of time, because of the long life cycle of trees. Overexpression of pine GS affected levels of GS activity, and the contents of chlorophyll and protein, which suggest that up-regulation of cytosolic GS1 may lead to a global effect on the synthesis of nitrogenous molecules in poplar leaves. In addition, these changes were associated with modification of the phenotype, and a correlation between GS activity in young leaves and the vegetative growth was found (J Fu, R Sampalo, F Gallardo, EG Kirby, FM Cánovas, unpublished data). Alterations of the phenotype include a higher leaf number and leaf surface area, which could explain the enhanced vegetative growth. These results suggest that the efficiency of N utilization may be engineered in trees by the manipulation of glutamine biosynthesis. Field trial studies have been initiated to evaluate the economic interest of the genetically modified poplar lines.
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Future prospects |
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TopAbstractIntroductionCharacteristics of GS, GOGAT...Expression studies as an...Modification of nitrogen...Future prospectsReferences |
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Considerable knowledge has been gained over the last ten years on the molecular characteristics and molecular regulation of N-assimilating enzymes in woody plants, including angiosperm and gymnosperm species. This research has greatly contributed to our understanding of how inorganic N is assimilated and utilized in trees. However, the available information is still limited and efforts should be made to increase basic research on N metabolism and to integrate new advances in biotechnology to improve growth and development of economically important woody species. Although all new studies will contribute to this goal, the concentration of efforts in model trees, such as poplar for angiosperms and pine for gymnosperms, is advisable.
In future years, the availability of new molecular tools for biological studies of trees will permit characterization of new genes involved in N metabolism and determination of their specific physiological roles. Functional studies are now possible in woody plants because routine transformation protocols via Agrobacterium are available for poplar and rapid progress has been reported in the last few years for conifers. The use of somatic embryogenic cell lines is critical in conifers for the generation of transgenic trees. Somatic embryogenesis also represents a useful model to study developmental gene expression and the functional roles of the enzymes during embryo development (Filonova et al., 2000). Results obtained with woody plant systems will be compared with information derived from structural/functional genomic studies in Arabidopsis and specific roles will be investigated in vivo by functional rescue of isolated mutants. For example, genomic technologies have recently been used to study the effect of a variety of N regimes on plant metabolism (Wang et al., 2000). Results from this study indicate that changes in N supply influence not only expression of genes involved in N assimilation, but also those involved in other metabolic pathways. Similar studies of gene expression at the organ or tissue levels are now feasible in tree models, with the existence of EST databases from poplar (http://www.forestbiotech.com) and loblolly and maritime pines (http://www.cbc.umn.edu; http://www.pierroton.inra.fr/Gemini). Studies on gene expression at the mRNA level will be complemented by proteomic analysis, including separation of proteins by 2D gel electrophoresis and sequence determination or mass spectrometry analysis of protease digestion products (Costa et al., 1999). Another promising line of research will be to study at the molecular level, the genetic basis of important traits, such as N use efficiency, grain yield, and height growth (Hirel et al., 2001). Genetic maps for poplar and pine have been established and now genes involved in N metabolism can be localized in the genome. The possible association of specific genes with quantitative trait loci (QTLs) are currently being investigated. The construction of defined BAC libraries for trees and the identification of individual clones using the isolated cDNAs and genomic clones as molecular probes, will accomplish the physical mapping of the regions of the genome where key genes are localized. This will allow molecular characterization of gene clusters involved in traits of interest in forestry and tree management.
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Acknowledgments |
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We would like to thank D Gifford for providing us unpublished material and EG Kirby for critical reading of the manuscript and valuable suggestions. Research work in the author's laboratory is funded by the Spanish Ministry of Science and Technology, Grants 1FD97-0746 and PB98-1396.
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Notes |
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1 To whom correspondence should be addressed. Fax: +34952132000. E-mail: canovas{at}uma.es
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References |
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TopAbstractIntroductionCharacteristics of GS, GOGAT...Expression studies as an...Modification of nitrogen...Future prospectsReferences |
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Aubert S, Bligny R, Douce R, Gout E, Ratcliffe RG, Roberts JKM. 2001. Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by 13C and 31P nuclear magnetic resonance. Journal of Experimental Botany 52, 37–45.
Avila C, Cantón FR, Barnestein P, Suárez MF, Marraccini P, Rey M, Humara J, Ordás R, Cánovas FM. 2001b. The promoter of a cytosolic glutamine synthetase gene from the conifer Pinus sylvestris is active in cotyledons of germinating seeds and light regulated in transgenic Arabidopsis thaliana. Physiologia Plantarum 112, 388–396.[Medline]
Avila C, García-Gutiérrez A, Crespillo R, Cánovas FM. 1998. Effects of phosphinothricin treatment on glutamine synthetase isoforms in Scots pine seedlings. Plant Physiology and Biochemistry 36, 857–863.[Web of Science]
Avila C, Muñoz-Chapuli R, Plomion C, Frigerio JM, Cánovas FM. 2000. Two genes encoding distinct cytosolic glutamine synthetases are closely linked in the pine genome. FEBS Letters 477, 237–243.[Web of Science][Medline]
Avila C, Suárez MF, Gómez-Maldonado J, Cánovas FM. 2001a. Spatial and temporal expression of two cytosolic glutamine synthetase genes in Scots pine: functional implications on nitrogen metabolism during early stages of conifer development. The Plant Journal 25, 93–102.[Web of Science][Medline]
Bauer D, Biehler K, Fock H, Carrayol E, Hirel B, Migge A, Becker TW. 1997. A role for cytosolic glutamine synthetase in the remobilization of leaf nitrogen during water stress in tomato. Physiologia Plantarum 99, 241–248.
Bedell J-P, Chalot M, Brun A, Botton B. 1995. Purification and properties of glutamine synthetase from Douglas-fir roots. Physiologia Plantarum 94, 597–604.
Bedell J-P, Chalot M, Garnier A, Botton B. 1999. Effects of nitrogen source on growth and activity of nitrogen-assimilating enzymes in Douglas-fir seedlings. Tree Physiology 19, 205–210.
Berg B. 1986. Nutrient release from litters and humus in coniferous forest soils. Scandinavian Journal Forest Research 1, 359–369.
Boiffin V, Hodges M, Gálvez S, Balestrini R, Bonfante P, Gadal P, Martin F. 1998. Eucalypt NADP+-dependent isocitrate dehydrogenase. cDNA cloning and expression in ectomycorrhizae. Plant Physiology 117, 939–944.
Brugière N, Dubois F, Limani A-ML, Roux Y, Sangwan RS, Hirel B. 1999. Glutamine synthetase in the phloem plays a major role in controlling proline production. The Plant Cell 11, 1995–2011.
Brugière N, Dubois F, Masclaux C, Sangwan RS, Hirel B. 2000. Immunolocalization of glutamine synthetase in senescing tobacco (Nicotiana tabacum L.) leaves suggests that ammonia assimilation is progresively shifted to the mesophyll cytosol. Planta 211, 519–527.[Web of Science][Medline]
Cánovas FM, Cantón FR, Gallardo F, García-Gutiérrez A, de Vicente A. 1991. Accumulation of glutamine synthetase during early development of maritime pine (Pinus pinaster) seedlings. Planta 185, 372–378.[Web of Science]
Cánovas FM, Cantón FR, García-Gutiérrez A, Gallardo F, Crespillo R. 1998. Molecular physiology of glutamine and glutamate biosynthesis in developing conifer seedlings. Physiologia Plantarum 103, 287–294.
Cantón FR, García-Gutiérrez A, Crespillo R, Cánovas FM. 1996. High-level expression of Pinus sylvestris glutamine synthetase in Escherichia coli. Production of polyclonal antibodies against the recombinant protein and expression studies in pine seedlings. FEBS Letters 393, 205–210.[Web of Science][Medline]
Cantón FR, García-Gutiérrez A, Gallardo F, de Vicente A, Cánovas FM. 1993. Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm: Pinus sylvestris. Plant Molecular Biology 22, 819–828.[Web of Science][Medline]
Cantón FR, Suárez M-F, José-Estanyol M, Cánovas FM. 1999. Expression analysis of a cytosolic glutamine synthetase gene in cotyledons of Scots pine seedlings: Developmental, light–dark regulation and spatial distribution of specific transcripts. Plant Molecular Biology 40, 623–634.[Web of Science][Medline]
Chen RD, Gadal P. 1990. Do the mitochondria provide the 2-oxoglutarate needed for glutamate synthesis in higher plants chloroplasts? Plant Physiology and Biochemistry 28, 141–145.[Web of Science]
Chevalier C, Bourgeois E, Just D, Raymond P. 1996. Metabolic regulation of asparagine synthetase gene expression in maize (Zea mays) root tips. The Plant Journal 9, 1–11.[Web of Science][Medline]
Coleman G, Bañados MP, Chen THH. 1994. Poplar bark storage protein and a related wound induced gene are differentially induced by nitrogen. Plant Physiology 106, 211–215.[Abstract]
Cornu S, Pireaux J-C, Gerard J, Dizengremel P. 1996. NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings. Physiologia Plantarum 96, 312–318.
Coschigano KT, Melo-Oliveira R, Lim J. 1998. Arabidopsis glts mutants and distinct Fdx-GOGAT genes: implications for photorespiration and primary nitrogen assimilation. The Plant Cell 10, 741–752.
Costa P, Pionneau C, Bauw G, Dubos C, Bahrman N, Kremer A, Frigerio J-M, Plomion C. 1999. Separation and characterization of needle and xylem maritime pine proteins. Electrophoresis 20, 1098–1108.[Web of Science][Medline]
Cren M, Hirel B. 1999. Glutamine synthetase in higher plants: regulation of gene and protein expression from the organ to the cell. Plant Cell Physiology 40, 1187–1193.
Dubois F, Brugière N, Sangwan RS, Hirel B. 1996. Localization of tobacco cytosolic glutamine synthetase enzymes and the corresponding transcripts shows organ- and cell-specific patterns of protein synthesis and gene expression. Plant Molecular Biology 31, 803–817.[Web of Science][Medline]
Edwards JW, Walker EL, Coruzzi GM. 1990. Cell-specific expression in transgenic plants reveals nonoverlapping roles for choroplast and cytosolic glutamine synthetase. Proceedings of National Academy of Sciences, USA 87, 3459–3463.
Elmlinger MV, Bolle C, Batschauer A, Oelmüller R, Mohr H. 1994. Coaction of blue light and light absorbed by phytochrome in control of glutamine synthetase gene expression in Scots pine (Pinus sylvestris L.) seedlings. Planta 192, 189–194.[Web of Science][Medline]
Elmlinger MV, Mohr H. 1991. Coaction of blue/ultraviolet-A light and light absorbed by phytochrome in controlling the appearance of ferredoxin-dependent glutamate synthase in the Scots pine (Pinus sylvestris L.) seedling. Planta 183, 374–380.[Web of Science]
Elmlinger MV, Mohr H. 1992. Glutamine synthetase in Scots pine seedlings and its control by blue light and light absorbed by phytochrome. Planta 188, 396–402.[Web of Science]
Filonova LH, Bozhkov PV, von Arnold S. 2000. Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking. Journal of Experimental Botany 51, 249–264.
Foyer CH, Ferrario S. 1994. Modulation of carbon and nitrogen metabolism in transgenic plants with a view to improved biomass production. Biochemical Society Transactions 22, 909–915.[Web of Science][Medline]
Gallardo F, Fu J, Cantón FR, García-Gutiérrez A, Cánovas FM, Kirby EG. 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19–26.[Web of Science][Medline]
Gálvez S, Lancien M, Hodges M. 1999. Are isocitrate dehydrogenases and 2-oxoglutarate involved in the regulation of glutamate biosynthesis? Trends in Plant Science 4, 484–490.[Web of Science][Medline]
García-Gutiérrez A, Cantón FR, Gallardo F, Sánchez-Jiménez F, Cánovas FM. 1995. Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings. Plant Molecular Biology 27, 115–128.[Web of Science][Medline]
García-Gutiérrez A, Dubois F, Cantón FR, Gallardo F, Sangwan RS, Cánovas FM. 1998. Two different modes of early development and nitrogen assimilation in gymnosperm seedlings. The Plant Journal 13, 187–199.[Web of Science]
García-Gutiérrez A, Gallardo F, Cantón FR, Crespillo R, Cánovas FM. 1996. Molecular analysis of pine ferredoxin-dependent glutamate synthase. In: Ahuja MR, Boerjan W, Neale D, eds. Somatic cell genetics and molecular genetics of trees. The Netherlands: Kluwer Academic Publishers, 189–195.
Guan C, Ribeiro A, Akkermans ADL, Jing Y, van Kammen A, Bisseling T, Pawlowski K. 1996. Nitrogen metabolism in actinorhizal nodules of Alnus glutinosa: expression of glutamine synthetase and acetylornithine transaminase. Plant Molecular Biology 32, 1177–1184.[Web of Science][Medline]
Hirel B, Bertin P, Quilleré I, Bourdoncle W, Attagnant C, Dellay C, Gouy A, Cadiou S, Retailleau C, Falque M, Gallais A. 2001. Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize. Plant Physiology 125, 1258–1270.
Hirel B, Perrot-Rechenmann C, Maudinas B, Gadal P. 1982. Glutamine synthetase in alder (Alnus glutinosa) root nodules. Purification, properties and cytoimmunochemical localization. Physiologia Plantarum 55, 197–203.
Hoshida H, Tanaka Y, Hibino T, Hayashi Y, Tanaka A, Takabe T, Takabe T. 2000. Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase. Plant Molecular Biology 43, 103–111.[Web of Science][Medline]
Ireland RJ, Lea PJ. 1999. The enzymes of glutamine, glutamate, asparagine, and aspartate metabolism. In: Singh BK, ed. Plant amino acids. Biochemistry and biotechnology. New York: Marcel Dekker Inc, 49–109.
Ishiyama K, Hayakawa T, Yamaya T. 1998. Expression of NADH-dependent glutamate synthase protein in the epidermis and exodermis of rice root in response to the supply of ammonium ions. Planta 204, 288–294.[Web of Science][Medline]
King JE, Gifford DG. 1997. Amino acid utilization in seeds of loblolly pine during germination and early seedling growth. Plant Physiology 113, 1124–1135.
Klopfenstein NB, Chum YW, Kim M-S, Ahuja MR. 1997. Micropropagation, genetic engineering and molecular biology of Populus. General Technical Report RM-GTR-297. Fort Collins: US Department of Agriculture, Forest Service, Rocky Mountain Forest and Range Experiment Station.
Kozaki A, Takeba G. 1996. Photorespiration protects C3 plants from photo-oxidation. Nature 384, 557–560.
Lam H-M, Koschigano KT, Oliveira IC, Melo-Oliveira R, Coruzzi GM. 1996. The molecular-genetics of nitrogen assimilation into amino acids in higher plants. Annual Review Plant Physiology and Plant Molecular Biology 47, 569–593.[Web of Science]
Limami A, Phillipson B, Ameziane R, Pernollet N, Jiang Q, Roy R, Deleens E, Chaumont-Bonnet M, Gresshoff PM, Hirel B. 1999. Does root glutamine synthetase control plant biomass production in Lotus japonicus L.? Planta 209, 495–502.[Web of Science][Medline]
Loulakakis KA, Roubelakis-Angelakis KA, Kanellis AK. 1994. Regulation of glutamate dehydrogenase and glutamine synthetase in avocado fruit during development and ripening. Plant Physiology 106, 217–222.[Abstract]
Loulakakis KA, Roubelakis-Angelakis KA. 1996. Characterization of Vitis vinifera L. glutamine synthetase and molecular cloning of cDNAs for the cytosolic enzyme. Plant Molecular Biology 31, 983–992.[Web of Science][Medline]
Loulakakis KA, Roubelakis-Angelakis KA. 1997. Molecular cloning and characterization of cDNAs coding for ferredoxin-dependent glutamate synthase from Vitis vinifera. Physiologia Plantarum 101, 220–228.
Ludwig RA. 1993. Arabidopsis chloroplasts dissimilate L-arginine and L-citrulline for use as nitrogen source. Plant Physiology 101, 429–434.[Abstract]
Martin F, Lorillou S. 1997. Nitrogen acquisition and assimilation in ectomycorrhizal systems. In: Renneberg H, Eschrich W, Ziegler H, eds. Trees—contributions to modern tree physiology. The Netherlands: Backhuys Publishers, 423–439.
Masclaux C, Valadier M-H, Brugière N, Morot-Gaudry J-F, Hirel B. 2000. Characterization of the sink/source transition in tobacco (Nicotiana tabacum L.) shoots in relation to nitrogen management and leaf senescence. Planta 211, 510–518.[Web of Science][Medline]
Melo-Oliveira R, Oliveira IC, Coruzzi M-G. 1996. Arabidopsis thaliana mutant analysis and gene regulation defines a non-redundant role for glutamate dehydrogenase in nitrogen assimilation. Proceedings of the National Academy of Sciences, USA 93, 4718–4723.
Miflin BJ, Lea PJ. 1980. Ammonia assimilation. In: Miflin BJ, ed. The biochemistry of plants, vol. 5. New York: Academic Press, 169–202.
Miflin BJ, Habash DZ. 2002. The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. Journal Experimental Botany 53, 979–987.
Migge A, Carrayol E, Hirel B, Becker TW. 2000. Leaf-specific overexpression of plastidic glutamine synthetase stimulates the growth of transgenic tobacco seedlings. Planta 210, 252–260.[Web of Science][Medline]
Nakano K, Suzuki T, Hayakawa T, Yamaya T. 2000. Organ and cellular localization of asparagine synthetase in rice plants. Plant Cell Physiology 41, 874–880.
Palomo J, Gallardo F, Suárez M-F, Cánovas FM. 1998. Purification and characterization of NADP+-linked isocitrate dehydrogenase from Scots pine. Evidence for different physiological roles of the enzyme in primary development. Plant Physiology 118, 617–626.
Pérez-García A, Cánovas FM, Gallardo F, Hirel B, de Vicente A. 1995. Differential expression of glutamine synthetase isoforms in tomato detached leaflets infected with Pseudomonas syringae pv. tomato. Molecular Plant–Microbe Interactions 8, 96–103.
Pérez-García A, de Vicente A, Cantón FR, Cazorla FM, Codina JC, García-Gutiérrez A, Cánovas FM. 1998a. Light-dependent changes of tomato glutamine synthetase in response to Pseudomonas syringae infection or phosphinothricin treatment. Physiologia Plantarum 102, 377–384.
Pérez-García A, Pereira S, Pisarra J, García-Gutiérrez A, Cazorla F, Salema R, de Vicente A, Cánovas FM. 1998b. Cytosolic localization in tomato mesophyll cells of a novel glutamine synthetase induced in response to bacterial infection or phosphinothricin treatment. Planta 206, 426–434.[Web of Science]
Pujade-Renaud V, Clement A, Perrot-Rechenmann C, Prévôt JC, Chrestin H, Jacob JL, Guern J. 1994. Ethylene induced increase in glutamine synthetase activity and in mRNA levels in Hevea brasiliensis latex cells. Plant Physiology 105, 127–132.[Abstract]
Pujade-Renaud V, Perrot-Rechenmann C, Chrestin H, Jacob JL, Guern J. 1997. Characterization of a full length cDNA clone encoding glutamine synthetase from ruber tree latex. Plant Physiology and Biochemistry 35, 85–93.[Web of Science]
Redinbaugh MG, Campbell WH. 1993. Glutamine synthetase and ferredoxin-dependent glutamate synthase expression in the maize (Zea mays) root primary response to nitrate. Evidence for an organ-specific response. Plant Physiology 101, 1249–1255.[Abstract]
Sakakibara H, Shimizu H, Hase T, Yamazaki Y, Takao T, Shimonishi Y, Sugiyama T. 1996. Molecular identification and characterization of cytosolic isoforms of glutamine synthetase in maize roots. Journal of Biological Chemistry 271, 29561–29568.
Schneider Wl, Gifford DJ. 1994. Loblolly pine seed dormancy. I. The relationship between protein synthesis and the loss of dormancy. Physiologia Plantarum 90, 246–252.
Sieciechowicz KA, Joy KW, Ireland RJ. 1988. The metabolism of asparagine in plants. Phytochemistry 27, 663–671.[Web of Science]
Simonson RL, Twigg P. 1999. Cloning and sequencing of a glutamine synthetase cDNA (Accession No. AF169795) from Juglans nigra (PGR99-144). Plant Physiology 121, 313.
Stewart GR, Shatilov VR, Turnbull MH, Robinson SA, Goodall R. 1995. Evidence that glutamate dehydrogenase plays a role in the oxidative deamination of glutamate in seedlings of Zea mays. Australian Journal Plant Physiology 22, 805–809.[Web of Science]
Schultz CJ, Hsu M, Miesak B, Coruzzi GM. 1998. Arabidopsis mutants define an in vivo role for isoenzymes of aspartate aminotransferase in plant nitrogen assimilation. Genetics 149, 491–499.
Temple SJ, Vance CP, Gantt JS. 1998. Glutamate synthase and nitrogen assimilation. Trends in Plant Science 3, 51–56.
Titus JS, Kang S-M. 1982. Nitrogen metabolism, translocation and recycling in apple trees. Horticultural Reviews 4, 204–246.
Trepp GB, Plank DW, Gantt S, Vance CP. 1999. NADH-glutamate synthase in alfalfa root nodules. Immunocytochemical localization. Plant Physiology 119, 829–837.
Todd CD, Cooke JEK, Mullen RT, Gifford DJ. 2001. Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth. Plant Molecular Biology 45, 555–565.[Web of Science][Medline]
Vézina LP, Margolis HA. 1990. Purification and properties of glutamine synthetase in leaves and roots of Pinus banksiana Lamb. Plant Physiology 94, 657–664.
Vincent R, Fraisier V, Chaillou S, Limani A, Deleens E, Phillipson B, Douat C, Boutin J-P, Hirel B. 1997. Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development. Planta 201, 424–433.[Web of Science][Medline]
Wang R, Guegler K, LaBrie ST, Crawford N. 2000. Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced. The Plant Cell 12, 1491–1509.
Woodall J, Forde BG. 1996. Glutamine synthetase polypeptides in the roots of 55 legume species in relation to their climatic origin an the partioning of nitrate assimilation. Plant, Cell and Environment 19, 848–858.
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