Rafael A. Cañas

High levels of asparagine synthetase in hypocotyls of pine seedlings suggest a role of the enzyme in re-allocation of seed-stored nitrogen

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.

Reprogramming of gene expression during compression wood formation in pine: Coordinated modulation of S-adenosylmethionine, lignin and lignan related genes

BackgroundTranscript 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.ResultsBy 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.ConclusionsThe 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.

Nitrogen metabolism in the developing ear of maize (Zea mays): analysis of two lines contrasting in their mode of nitrogen management.

*The main steps of nitrogen (N) metabolism were characterized in the developing ear of the two maize (Zea mays) lines F2 and Io, which were previously used to investigate the genetic basis of nitrogen use efficiency (NUE) in relation to yield. *During the grain-filling period, we monitored changes in metabolite content, enzyme activities and steady-state levels of transcripts for marker genes of amino acid synthesis and interconversion in the cob and the kernels. *Under low N fertilization conditions, line Io accumulated glutamine, asparagine and alanine preferentially in the developing kernels, whereas in line F2, glutamine and proline were the predominant amino acids. Quantification of the mRNA-encoding enzymes involved in asparagine, alanine and proline biosynthesis confirmed that the differences observed between the two lines at the physiological level are likely to be attributable to enhanced expression of the cognate genes. *Integrative analysis of physiological and gene expression data indicated that the developing ear of line Io had higher N use and transport capacities than line F2. Thus, in maize there is genetic and environmental control of N metabolism not only in vegetative source organs but also in reproductive sink organs.

Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants

In the chloroplasts and in non-green plastids of plants, aspartate is the precursor for the biosynthesis of different amino acids and derived metabolites that play distinct and important roles in plant growth, reproduction, development or defence. Aspartate biosynthesis is mediated by the enzyme aspartate aminotransferase (EC 2.6.1.1), which catalyses the reversible transamination between glutamate and oxaloacetate to generate aspartate and 2-oxoglutarate. Plastids contain two aspartate aminotransferases: a eukaryotic-type and a prokaryotic-type bifunctional enzyme displaying aspartate and prephenate aminotransferase activities. A general overview of the biochemistry, regulation, functional significance, and phylogenetic origin of both enzymes is presented. The roles of these plastidic aminotransferases in the biosynthesis of essential amino acids are discussed.

Coordination of PsAS1 and PsASPG expression controls timing of re-allocated N utilization in hypocotyls of pine seedlings

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.

Understanding developmental and adaptive cues in pine through metabolite profiling and co-expression network analysis

Highlight This work highlights the complex interaction between developmental processes and environmental adaptations of maritime pine trees growing under natural conditions.

Transcriptome analysis in maritime pine using laser capture microdissection and 454 pyrosequencing

Maritime pine (Pinus pinaster Aiton) is one of the most advanced conifer models for genomics research. Conifer genomes are extremely large and major advances have recently been made in the characterization of transcriptomes. The combination of laser capture microdissection (LCM) and next-generation sequencing is a powerful tool with which to resolve the entire transcriptome of specific cell types and tissues. In the current work, we have developed a protocol for transcriptomic analyses of conifer tissue types using LCM and 454 pyrosequencing. Tissue sections were isolated using non-fixed flash-frozen samples processed by LCM. Complementary DNA synthesis and amplification from tiny amounts of total RNA from LCM samples was performed using an adapted protocol for C: onifer R: NA A: mplification (CRA+). The cDNA amplification yield and cDNA quality provided by CRA+ were adequate for 454 pyrosequencing. Furthermore, read length and quality results of the 454 runs were near the optimal parameters considered by Roche for transcriptome sequencing. Using the CRA+ protocol, non-specific amplifications were prevented, problems derived from poly(A:T) tails in the 454 sequencing technology were reduced, and read length and read number considerably enhanced. This technical approach will facilitate global gene expression analysis in individual tissues of conifers and may also be applied to other plant species.

Can genetic variability for nitrogen metabolism in the developing ear of maize be exploited to improve yield

Quantitative trait loci (QTLs) for the main steps of nitrogen (N) metabolism in the developing ear of maize (Zea mays L.) and their co-localization with QTLs for kernel yield and putative candidate genes were searched in order to identify chromosomal regions putatively involved in the determination of yield. During the grain-filling period, the changes in physiological traits were monitored in the cob and in the developing kernels, representative of carbon and N metabolism in the developing ear. The correlations between these physiological traits and traits related to yield were examined and localized with the corresponding QTLs on a genetic map. Glycine and serine metabolism in developing kernels and the cognate genes appeared to be of major importance for kernel production. The importance of kernel glutamine synthesis in the determination of yield was also confirmed. The genetic and physiological bases of N metabolism in the developing ear can be studied in an integrated manner by means of a quantitative genetic approach using molecular markers and genomics, and combining agronomic, physiological and correlation studies. Such an approach leads to the identification of possible new regulatory metabolic and developmental networks specific to the ear that may be of major importance for maize productivity.

Molecular Modeling and Site-Directed Mutagenesis Reveal Essential Residues for Catalysis in a Prokaryote-Type Aspartate Aminotransferase

We recently reported that aspartate (Asp) biosynthesis in plant chloroplasts is catalyzed by two different Asp aminotransferases (AAT): a previously characterized eukaryote type and a prokaryote type (PT-AAT) similar to bacterial and archaebacterial enzymes. The available molecular and kinetic data suggest that the eukaryote-type AAT is involved in the shuttling of reducing equivalents through the plastidic membrane, whereas the PT-AAT could be involved in the biosynthesis of the Asp-derived amino acids inside the organelle. In this work, a comparative modeling of the PT-AAT enzyme from Pinus pinaster (PpAAT) was performed using x-ray structures of a bacterial AAT (Thermus thermophilus; Protein Data Bank accession nos. 1BJW and 1BKG) as templates. We computed a three-dimensional folding model of this plant homodimeric enzyme that has been used to investigate the functional importance of key amino acid residues in its active center. The overall structure of the model is similar to the one described for other AAT enzymes, from eukaryotic and prokaryotic sources, with two equivalent active sites each formed by residues of both subunits of the homodimer. Moreover, PpAAT monomers folded into one large and one small domain. However, PpAAT enzyme showed unique structural and functional characteristics that have been specifically described in the AATs from the prokaryotes Phormidium lapideum and T. thermophilus, such as those involved in the recognition of the substrate side chain or the “open-to-closed” transition following substrate binding. These predicted characteristics have been substantiated by site-direct mutagenesis analyses, and several critical residues (valine-206, serine-207, glutamine-346, glutamate-210, and phenylalanine-450) were identified and functionally characterized. The reported data represent a valuable resource to understand the function of this enzyme in plant amino acid metabolism.

Biosynthesis and Metabolic Fate of Phenylalanine in Conifers.

The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions, and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined.