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Epitranscriptome constitutes a gene expression checkpoint in all living organisms. Nitrogen is an essential element for plant growth and development that influences gene expression at different levels such as epigenome, transcriptome, proteome, and metabolome. Therefore, our hypothesis is that changes in the epitranscriptome may regulate nitrogen metabolism. In this study, epitranscriptomic modifications caused by ammonium nutrition were monitored in maritime pine roots using Oxford Nanopore Technology. Transcriptomic responses mainly affected transcripts involved in nitrogen and carbon metabolism, defense, hormone synthesis/signaling, and translation. Global detection of epitranscriptomic marks was performed to evaluate this posttranscriptional mechanism in un/treated seedlings. Increased N 6 -methyladenosine (m 6 A) deposition in the 3'-UTR was observed in response to ammonium, which seems to be correlated with poly(A) lengths and changes in the relative abundance of the corresponding proteins. The results showed that m 6 A deposition and its dynamics seem to be important regulators of translation under ammonium nutrition. These findings suggest that protein translation is finely regulated through epitranscriptomic marks likely by changes in mRNA poly(A) length, transcript abundance and ribosome protein composition. An integration of multiomics data suggests that the epitranscriptome modulates responses to nutritional, developmental and environmental changes through buffering, filtering, and focusing the final products of gene expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ortigosa, Lobato-Fernández, Pérez-Claros, Cantón, Ávila, Cánovas and Cañas.)

Glutamine synthetase (GS) is a key enzyme responsible for the incorporation of inorganic nitrogen in the form of ammonium into the amino acid glutamine. In plants, two groups of functional GS enzymes are found: eubacterial GSIIb (GLN2) and eukaryotic GSIIe (GLN1/GS). Only GLN1/GS genes are found in vascular plants, which suggests that they are involved in the final adaptation of plants to terrestrial life. The present phylogenetic study reclassifies the different GS genes of seed plants into three clusters: GS1a, GS1b and GS2. The presence of genes encoding GS2 has been expanded to Cycadopsida gymnosperms, which suggests the origin of this gene in a common ancestor of Cycadopsida, Ginkgoopsida and angiosperms. GS1a genes have been identified in all gymnosperms, basal angiosperms and some Magnoliidae species. Previous studies in conifers and the gene expression profiles obtained in ginkgo and magnolia in the present work could explain the absence of GS1a in more recent angiosperm species (e.g. monocots and eudicots) as a result of the redundant roles of GS1a and GS2 in photosynthetic cells. Altogether, the results provide a better understanding of the evolution of plant GS isoenzymes and their physiological roles, which is valuable for improving crop nitrogen use efficiency and productivity. This new view of GS evolution in plants, including a new cytosolic GS group (GS1a), has important functional implications in the context of plant metabolism adaptation to global changes., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Motivation: The oxidation of protein-bound methionine to form methionine sulfoxide has traditionally been regarded as an oxidative damage. However, growing evidences support the view of this reversible reaction also as a regulatory post-translational modification. Thus, the oxidation of methionine residues has been reported to have multiple and varied implications for protein function. However, despite the importance of this modification and the abundance of reports, all these data are scattered in the literature. No database/resource on methionine sulfoxidation exists currently. Since this information is useful to gain further insights into the redox regulation of cellular proteins, we have created a primary database of experimentally confirmed sulfoxidation sites., Results: MetOSite currently contains 7242 methionine sulfoxide sites found in 3562 different proteins from 23 species, with Homo sapiens, Arabidopsis thaliana and Bacillus cereus as the main contributors. Each collected site has been classified according to the effect of its sulfoxidation on the biological properties of the modified protein. Thus, MetOSite documents cases where the sulfoxidation of methionine leads to (i) gain of activity, (ii) loss of activity, (iii) increased protein-protein interaction susceptibility, (iv) decreased protein-protein interaction susceptibility, (v) changes in protein stability and (vi) changes in subcellular location., Availability and Implementation: MetOSite is available at https://metosite.uma.es., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The oxidation of protein-bound methionine to form methionine sulfoxide, has traditionally been regarded as an oxidative damage. However, recent evidences support the view of this reversible reaction as a regulatory post-translational modification. The perception that methionine sulfoxidation may provide a mechanism to the redox regulation of a wide range of cellular processes, has stimulated some proteomic studies. However, these experimental approaches are expensive and time-consuming. Therefore, computational methods designed to predict methionine oxidation sites are an attractive alternative. As a first approach to this matter, we have developed models based on random forests, support vector machines and neural networks, aimed at accurate prediction of sites of methionine oxidation., Results: Starting from published proteomic data regarding oxidized methionines, we created a hand-curated dataset formed by 113 unique polypeptides of known structure, containing 975 methionyl residues, 122 of which were oxidation-prone (positive dataset) and 853 were oxidation-resistant (negative dataset). We use a machine learning approach to generate predictive models from these datasets. Among the multiple features used in the classification task, some of them contributed substantially to the performance of the predictive models. Thus, (i) the solvent accessible area of the methionine residue, (ii) the number of residues between the analyzed methionine and the next methionine found towards the N-terminus and (iii) the spatial distance between the atom of sulfur from the analyzed methionine and the closest aromatic residue, were among the most relevant features. Compared to the other classifiers we also evaluated, random forests provided the best performance, with accuracy, sensitivity and specificity of 0.7468±0.0567, 0.6817±0.0982 and 0.7557±0.0721, respectively (mean ± standard deviation)., Conclusions: We present the first predictive models aimed to computationally detect methionine sites that may become oxidized in vivo in response to oxidative signals. These models provide insights into the structural context in which a methionine residue become either oxidation-resistant or oxidation-prone. Furthermore, these models should be useful in prioritizing methinonyl residues for further studies to determine their potential as regulatory post-translational modification sites.

Asparaginases (ASPG, EC 3.5.1.1) catalyze the hydrolysis of the amide group of L-asparagine producing L-aspartate and ammonium. Three ASPG, PpASPG1, PpASPG2, and PpASPG3, have been identified in the transcriptome of maritime pine ( Pinus pinaster Ait.) that were transiently expressed in Nicotiana benthamiana by agroinfection. The three recombinant proteins were processed in planta to active enzymes and it was found that all mature forms exhibited double activity asparaginase/isoaspartyl dipeptidase but only PpASPG1 was able to catalyze efficiently L-asparagine hydrolysis. PpASPG1 contains a variable region of 77 amino acids that is critical for proteolytic processing of the precursor and is retained in the mature enzyme. Furthermore, the functional analysis of deletion mutants demonstrated that this protein fragment is required for specific recognition of the substrate and favors enzyme stability. Potassium has a limited effect on the activation of maritime pine ASPG what is consistent with the lack of a critical residue essential for interaction of cation. Taken together, the results presented here highlight the specific features of ASPG from conifers when compared to the enzymes from angiosperms.

Protein phosphorylation is one of the most prevalent and well-understood protein modifications. Oxidation of protein-bound methionine, which has been traditionally perceived as an inevitable damage derived from oxidative stress, is now emerging as another modification capable of regulating protein activity during stress conditions. However, the mechanism coupling oxidative signals to changes in protein function remains unknown. An appealing hypothesis is that methionine oxidation might serve as a rheostat to control phosphorylation. To investigate this potential crosstalk between phosphorylation and methionine oxidation, we have addressed the co-occurrence of these two types of modifications within the human proteome. Here, we show that nearly all (98%) proteins containing oxidized methionine were also phosphoproteins. Furthermore, phosphorylation sites were much closer to oxidized methionines when compared to non-oxidized methionines. This proximity between modification sites cannot be accounted for by their co-localization within unstructured clusters because it was faithfully reproduced in a smaller sample of structured proteins. We also provide evidence that the oxidation of methionine located within phosphorylation motifs is a highly selective process among stress-related proteins, which supports the hypothesis of crosstalk between methionine oxidation and phosphorylation as part of the cellular defence against oxidative stress.

Methionine residues exhibit different degrees of susceptibility to oxidation. Although solvent accessibility is a relevant factor, oxidation at particular sites cannot be unequivocally explained by accessibility alone. To explore other possible structural determinants, we assembled different sets of oxidation-sensitive and oxidation-resistant methionines contained in human proteins. Comparisons of the proteins containing oxidized methionines with all proteins in the human proteome led to the conclusion that the former exhibit a significantly higher mean value of methionine content than the latter. Within a given protein, an examination of the sequence surrounding the non-oxidized methionine revealed a preference for neighbouring tyrosine and tryptophan residues, but not for phenylalanine residues. However, because the interaction between sulphur atoms and aromatic residues has been reported to be important for the stabilization of protein structure, we carried out an analysis of the spatial interatomic distances between methionines and aromatic residues, including phenylalanine. The results of these analyses uncovered a new determinant for methionine oxidation: the S-aromatic motif, which decreases the reactivity of the involved sulphur towards oxidants.

Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Transcript profiling of differentiating secondary xylem has allowed us to draw a general picture of the genes involved in wood formation. However, our knowledge is still limited about the regulatory mechanisms that coordinate and modulate the different pathways providing substrates during xylogenesis. The development of compression wood in conifers constitutes an exceptional model for these studies. Although differential expression of a few genes in differentiating compression wood compared to normal or opposite wood has been reported, the broad range of features that distinguish this reaction wood suggest that the expression of a larger set of genes would be modified., Results: By combining the construction of different cDNA libraries with microarray analyses we have identified a total of 496 genes in maritime pine (Pinus pinaster, Ait.) that change in expression during differentiation of compression wood (331 up-regulated and 165 down-regulated compared to opposite wood). Samples from different provenances collected in different years and geographic locations were integrated into the analyses to mitigate the effects of multiple sources of variability. This strategy allowed us to define a group of genes that are consistently associated with compression wood formation. Correlating with the deposition of a thicker secondary cell wall that characterizes compression wood development, the expression of a number of genes involved in synthesis of cellulose, hemicellulose, lignin and lignans was up-regulated. Further analysis of a set of these genes involved in S-adenosylmethionine metabolism, ammonium recycling, and lignin and lignans biosynthesis showed changes in expression levels in parallel to the levels of lignin accumulation in cells undergoing xylogenesis in vivo and in vitro., Conclusions: The comparative transcriptomic analysis reported here have revealed a broad spectrum of coordinated transcriptional modulation of genes involved in biosynthesis of different cell wall polymers associated with within-tree variations in pine wood structure and composition. In particular, we demonstrate the coordinated modulation at transcriptional level of a gene set involved in S-adenosylmethionine synthesis and ammonium assimilation with increased demand for coniferyl alcohol for lignin and lignan synthesis, enabling a better understanding of the metabolic requirements in cells undergoing lignification.

Background: Pinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases., Description: EuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided., Conclusions: The EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.

Background: Multiple sequence alignments are used to study gene or protein function, phylogenetic relations, genome evolution hypotheses and even gene polymorphisms. Virtually without exception, all available tools focus on conserved segments or residues. Small divergent regions, however, are biologically important for specific quantitative polymerase chain reaction, genotyping, molecular markers and preparation of specific antibodies, and yet have received little attention. As a consequence, they must be selected empirically by the researcher. AlignMiner has been developed to fill this gap in bioinformatic analyses., Results: AlignMiner is a Web-based application for detection of conserved and divergent regions in alignments of conserved sequences, focusing particularly on divergence. It accepts alignments (protein or nucleic acid) obtained using any of a variety of algorithms, which does not appear to have a significant impact on the final results. AlignMiner uses different scoring methods for assessing conserved/divergent regions, Entropy being the method that provides the highest number of regions with the greatest length, and Weighted being the most restrictive. Conserved/divergent regions can be generated either with respect to the consensus sequence or to one master sequence. The resulting data are presented in a graphical interface developed in AJAX, which provides remarkable user interaction capabilities. Users do not need to wait until execution is complete and can.even inspect their results on a different computer. Data can be downloaded onto a user disk, in standard formats. In silico and experimental proof-of-concept cases have shown that AlignMiner can be successfully used to designing specific polymerase chain reaction primers as well as potential epitopes for antibodies. Primer design is assisted by a module that deploys several oligonucleotide parameters for designing primers "on the fly"., Conclusions: AlignMiner can be used to reliably detect divergent regions via several scoring methods that provide different levels of selectivity. Its predictions have been verified by experimental means. Hence, it is expected that its usage will save researchers' time and ensure an objective selection of the best-possible divergent region when closely related sequences are analysed. AlignMiner is freely available at http://www.scbi.uma.es/alignminer.

Background: High-throughput automated sequencing has enabled an exponential growth rate of sequencing data. This requires increasing sequence quality and reliability in order to avoid database contamination with artefactual sequences. The arrival of pyrosequencing enhances this problem and necessitates customisable pre-processing algorithms., Results: SeqTrim has been implemented both as a Web and as a standalone command line application. Already-published and newly-designed algorithms have been included to identify sequence inserts, to remove low quality, vector, adaptor, low complexity and contaminant sequences, and to detect chimeric reads. The availability of several input and output formats allows its inclusion in sequence processing workflows. Due to its specific algorithms, SeqTrim outperforms other pre-processors implemented as Web services or standalone applications. It performs equally well with sequences from EST libraries, SSH libraries, genomic DNA libraries and pyrosequencing reads and does not lead to over-trimming., Conclusions: SeqTrim is an efficient pipeline designed for pre-processing of any type of sequence read, including next-generation sequencing. It is easily configurable and provides a friendly interface that allows users to know what happened with sequences at every pre-processing stage, and to verify pre-processing of an individual sequence if desired. The recommended pipeline reveals more information about each sequence than previously described pre-processors and can discard more sequencing or experimental artefacts.

We report the molecular characterization and functional analysis of a gene (PsdeltaOAT) from Scots pine (Pinus sylvestris) encoding Orn-delta-aminotransferase (delta-OAT; EC 2.6.1.13), an enzyme of arginine metabolism. The deduced amino acid sequence contains a putative N-terminal signal peptide for mitochondrial targeting. The polypeptide is similar to other delta-OATs from plants, yeast, and mammals and encoded by a single-copy gene in pine. PsdeltaOAT encodes a functional delta-OAT as determined by expression of the recombinant protein in Escherichia coli and analysis of the active enzyme. The expression of PsdeltaOAT was undetectable in the embryo, but highly induced at early stages of germination and seedling development in all different organs. Transcript levels decreased in later developmental stages, although an increase was observed in lignified stems of 90-d-old plants. An increase of delta-OAT activity was observed in germinating embryos and seedlings and appears to mirror the observed alterations in PsdeltaOAT transcript levels. Similar expression patterns were also observed for genes encoding arginase and isocitrate dehydrogenase. Transcripts of PsdeltaOAT and the arginase gene were found widely distributed in different cell types of pine organs. Consistent with these results a metabolic pathway is proposed for the nitrogen flow from the megagametophyte to the developing seedling, which is also supported by the relative abundance of free amino acids in embryos and seedlings. Taken together, our data support that delta-OAT plays an important role in this process providing glutamate for glutamine biosynthesis during early pine growth.

As part of a study aimed at understanding the physiological and molecular mechanisms involved in adventitious shoot bud formation in pine cotyledons, we conducted a transcriptome analysis to identify early-induced genes during the first phases of adventitious caulogenesis in Pinus pinea L. cotyledons cultured in the presence of benzyladenine. A subtractive cDNA library with more than 700 clones was constructed. Of these clones, 393 were sequenced, analyzed and grouped according to their putative function. Quantitative real-time PCR analysis was performed to confirm the differential expression of 30 candidate genes. Results are contrasted with available data for other species.

During pine seed germination, a large amount of N mobilized from the storage proteins is re-allocated in the hypocotyl as free asparagine, as a result of the high levels of asparagine synthetase (AS) encoded by the PsAS1 gene. To determine the role of this re-allocated N reserve, a full-length cDNA encoding L: -asparaginase (ASPG) has been cloned from Scots pine (Pinus sylvestris L.) seedlings and characterized. Like other N-terminal nucleophile hydrolases, pine ASPG requires a post-translational processing to exhibit enzymatic activity. However, in contrast to previous reports on other plant ASPGs, purified recombinant pine ASPG does not undergo autoproteolytic cleavage in vitro. Our results suggest that the processing requires accessory proteins to assist in the proteolysis or in the proper folding before autocleavage in a divalent cation-dependent manner. Sequence comparison analysis revealed that the pine protein is included in the K+-dependent subfamily of plant ASPGs. The expression of the ASPG-encoding gene (PsASPG) was higher in organs with extensive secondary development of the vascular system. The increase in transcript abundance observed at advanced stages of hypocotyl development was concomitant with a decrease of PsAS1 transcript abundance and a remarkable increase in the number of xylem elements and highly lignified cell walls. These results, together with the precise localization of PsASPG transcripts in cells of the cambial region, suggest that the expression of PsAS1 and PsASPG is temporally coordinated, to control the re-allocation of N from seed storage proteins toward the hypocotyl to be later used during early development of secondary vascular system.

Conifers are the most important group of gymnosperms, which include tree species of great ecological and economic importance that dominate large ecosystems and play an essential role in global carbon fixation. Nitrogen (N) economy has a special importance in these woody plants that are able to cope with seasonal periods of growth and development over a large number of years. As N availability in the forest soil is extremely low, efficient mechanisms are required for the assimilation, storage, mobilization, and recycling of inorganic and organic forms of N. The cyclic interconversion of arginine and the amides glutamine and asparagine plays a central role in the N metabolism of conifers and the regulation of these pathways is of major relevance to the N economy of the plant. In this paper, details of recent progress in our understanding of the metabolism of arginine and the other major amino acids glutamine, glutamate, aspartate, and asparagine in pine, a conifer model tree, are presented and discussed.

A pine asparagine synthetase gene expressed in developing seedlings has been identified by cloning its cDNA (PsAS1) from Scots pine (Pinus sylvestris L.). Genomic DNA analysis with PsAS1 probes and a sequence-based phylogenetic tree are consistent with the possibility of more than one gene encoding asparagine synthetase in pine. However, the parallel patterns of free asparagine content and PsAS1 products indicate that the protein encoded by this gene is mainly responsible for the accumulation of this amino acid during germination and early seedling development. The temporal and spatial patterns of PsAS1 expression together with the spatial distribution of asparagine content suggest that, early after germination, part of the nitrogen mobilized from the megagametophyte is diverted toward the hypocotyl to produce high levels of asparagine as a reservoir of nitrogen to meet later specific demands of development. Furthermore, the transcript and protein analyses in seedlings germinated and growth for extended periods under continuous light or dark suggest that the spatial expression pattern of PsAS1 is largely determined by a developmental program. Therefore, our results suggest that the spatial and temporal control of PsAS1 expression determines the re-allocation of an important amount of seed-stored nitrogen during pine germination.

An enzymatically active recombinant protein kinase, previously isolated and characterized in Fagus sylvatica L. dormant seeds (FsPK1), was used to obtain a specific polyclonal antibody against this protein. Immunoblotting and immunohistochemical analysis of FsPK1 protein in beech seeds showed a strong immunostaining in the nucleus of the cells located in the vascular tissue of the embryonic axis corresponding to the future apical meristem of the root. This protein kinase was found to accumulate in the seeds only when embryo growth was arrested by application of ABA, while the protein amount decreased during stratification, previously proved to alleviate dormancy, and no protein was detected at all when seed germination was induced by addition of GA(3). These results indicate that FsPK1 may be involved in the control of the embryo growth mediated by ABA and GAs during the transition from dormancy to germination in Fagus sylvatica seeds.

Plants have developed a variety of molecular strategies to use limiting nutrients with a maximum efficiency. N assimilated into biomolecules can be released in the form of ammonium by plant metabolic activities in various physiological processes such as photorespiration, the biosynthesis of phenylpropanoids or the mobilization of stored reserves. Thus, efficient reassimilation mechanisms are required to reincorporate liberated ammonium into metabolism and maintain N plant economy. Although the biochemistry and molecular biology of ammonium recycling in annual herbaceous plants has been previously reported, the recent advances in woody plants need to be reviewed. Moreover, it is important to point out that N recycling is quantitatively massive during some of these metabolic processes in trees, including seed germination, the onset of dormancy and resumption of active growth or the biosynthesis of lignin that takes place during wood formation. Therefore, woody plants constitute an excellent system as a model to study N mobilization and recycling. The aim of this paper is to provide an overview of different physiological processes in woody perennials that challenge the overall plant N economy by releasing important amounts of inorganic N in the form of ammonium.

Nitrogen metabolism is one aspect of basic metabolism, which is still quite unknown in the field of plant-pathogen interactions. Evidence derived from previous studies conducted in our laboratory strongly suggests that during microbial pathogenesis an important nitrogen mobilization process takes place in diseased tissues. Here we describe the expression pattern of asparagine synthetase (AS; EC 6.3.5.4) in tomato leaves infected by the bacterial pathogen Pseudomonas syringae pv. tomato. Using an homologous AS cDNA probe isolated by RT-PCR from infected leaves, we have observed a high level induction of AS expression during the course of infection. Concomitantly, a single AS polypeptide also accumulated in response to bacterial infection. Furthermore, immunohistochemical analysis of AS in infected leaves revealed a strong immunostaining in phloem cells of the main vascular bundles and in secondary veins of the leaf blade. These data correlate with those previously reported for expression of a cytosolic isoform of glutamine synthetase (GS1) also induced during development of the infectious process. Taken together, our results suggest the existence of a GS1/AS pathway representing a metabolic route for transferring ammonium released from protein catabolism into asparagine, an amino acid that may have a major role in nitrogen mobilization from diseased tissues.

Two glutamine synthetase isogenes, GS1a and GS1b, isolated from pine have been functionally expressed in E. coli and the characteristics of individual gene products compared. When bacteria were grown at 37 degrees C most pine GS1 protein was found in the insoluble fraction but lowering of the expression temperature increased yield of both GS1 polypeptide and activity in the soluble fraction. High levels of functionally active GS1a (309 + or - 35 nkat mg(-1)) and GS1b (1,166 + or - 65 nkat mg(-1)) enzymes were obtained by decreasing the expression temperature to 10 degrees C. Purification and characterization of recombinant products showed that pine GS1 polypeptides are assembled in octameric GS holoenzymes showing structural and kinetic differences. The results are discussed with regard to the specific localization of GS1a and GS1b in different cell types of pine seedlings. The isoform GS1a may control the assimilation of the high levels of ammonium released in photosynthetic tissues, whereas GS1b enzyme could mitigate oscillations in glutamate availability providing a constant flux of glutamine for nitrogen transport in vascular cells.

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.