Pine has two glutamine synthetase paralogs, GS1b.1 and GS1b.2, exhibiting distinct biochemical properties.

The enzyme glutamine synthetase (EC 6.3.1.2) is mainly responsible for the incorporation of inorganic nitrogen into organic molecules in plants. In the present work, a pine (Pinus pinaster) GS1 (PpGS1b.2) gene was identified, showing a high sequence identity with the GS1b.1 gene previously characterized in conifers. Phylogenetic analysis revealed that the presence of PpGS1b.2 is restricted to the genera Pinus and Picea and is not found in other conifers. Gene expression data suggest a putative role of PpGS1b.2 in plant development, similar to other GS1b genes from angiosperms, suggesting evolutionary convergence. The characterization of GS1b.1 and GS1b.2 at the structural, physicochemical, and kinetic levels has shown differences even though they have high sequence homology. GS1b.2 had a lower optimum pH (6 vs. 6.5) and was less thermally stable than GS1b.1. GS1b.2 exhibited positive cooperativity for glutamate and substrate inhibition for ammonium. However, GS1b.1 exhibited substrate inhibition behavior for glutamate and ATP. Alterations in the kinetic characteristics produced by site-directed mutagenesis carried out in this work strongly suggest an implication of amino acids at positions 264 and 267 in the active center of pine GS1b.1 and GS1b.2 being involved in affinity toward ammonium. Therefore, the amino acid differences between GS1b.1 and GS1b.2 would support the functioning of both enzymes to meet distinct plant needs.

Transcriptome Analysis and Intraspecific Variation in Spanish Fir (Abies pinsapo Boiss.).

Spanish fir (Abies pinsapo Boiss.) is an endemic, endangered tree that has been scarcely investigated at the molecular level. In this work, the transcriptome of Spanish fir was assembled, providing a large catalog of expressed genes (22,769), within which a high proportion were full-length transcripts (12,545). This resource is valuable for functional genomics studies and genome annotation in this relict conifer species. Two intraspecific variations of A. pinsapo can be found within its largest population at the Sierra de las Nieves National Park: one with standard green needles and another with bluish-green needles. To elucidate the causes of both phenotypes, we studied different physiological and molecular markers and transcriptome profiles in the needles. "Green" trees showed higher electron transport efficiency and enhanced levels of chlorophyll, protein, and total nitrogen in the needles. In contrast, needles from "bluish" trees exhibited higher contents of carotenoids and cellulose. These results agreed with the differential transcriptomic profiles, suggesting an imbalance in the nitrogen status of "bluish" trees. Additionally, gene expression analyses suggested that these differences could be associated with different epigenomic profiles. Taken together, the reported data provide new transcriptome resources and a better understanding of the natural variation in this tree species, which can help improve guidelines for its conservation and the implementation of adaptive management strategies under climatic change.

Is plastidic glutamine synthetase essential for C3 plants? A tale of photorespiratory mutants, ammonium tolerance and conifers.

Agriculture faces the considerable challenge of having to adapt to a progressively changing climate (including the increase in CO2 levels and temperatures); environmental impact must be reduced while at the same time crop yields need to be maintained or increased to ensure food security. Under this scenario, increasing plants' nitrogen (N) use efficiency and minimizing the energy losses associated with photorespiration are two goals of crop breeding that are long sought after. The plastidic glutamine synthetase (GS2) enzyme stands at the crossroads of N assimilation and photorespiration, and is therefore a key candidate for the improvement of crop performance. The GS2 enzyme has long been considered essential for angiosperm survival under photorespiratory conditions. Surprisingly, in Arabidopsis GS2 is not essential for plant survival, and its absence confers tolerance towards ammonium stress, which is in conflict with the idea that NH4+ accumulation is one of the main causes of ammonium stress. Altogether, it appears that the 'textbook' view of this enzyme must be revisited, especially regarding the degree to which it is essential for plant growth under photorespiratory conditions, and the role of NH4+ assimilation during ammonium stress. In this article we open the debate on whether more or less GS2 is a desirable trait for plant productivity.

A revised view on the evolution of glutamine synthetase isoenzymes in plants.

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.

Ammonium regulates the development of pine roots through hormonal crosstalk and differential expression of transcription factors in the apex.

Ammonium is a prominent source of inorganic nitrogen for plant nutrition, but excessive amounts can be toxic for many species. However, most conifers are tolerant to ammonium, a relevant physiological feature of this ancient evolutionary lineage. For a better understanding of the molecular basis of this trait, ammonium-induced changes in the transcriptome of maritime pine (Pinus pinaster Ait.) root apex have been determined by laser capture microdissection and RNA sequencing. Ammonium promoted changes in the transcriptional profiles of multiple transcription factors, such as SHORT-ROOT, and phytohormone-related transcripts, such as ACO, involved in the development of the root meristem. Nano-PALDI-MSI and transcriptomic analyses showed that the distributions of IAA and CKs were altered in the root apex in response to ammonium nutrition. Taken together, the data suggest that this early response is involved in the increased lateral root branching and principal root growth, which characterize the long-term response to ammonium supply in pine. All these results suggest that ammonium induces changes in the root system architecture through the IAA-CK-ET phytohormone crosstalk and transcriptional regulation.

Epitranscriptome changes triggered by ammonium nutrition regulate the proteome response of maritime pine roots.

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 N6-methyladenosine (m6A) 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 m6A 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.

Inorganic Nitrogen Form Determines Nutrient Allocation and Metabolic Responses in Maritime Pine Seedlings.

Nitrate and ammonium are the main forms of inorganic nitrogen available to plants. The present study aimed to investigate the metabolic changes caused by ammonium and nitrate nutrition in maritime pine (Pinus pinaster Ait.). Seedlings were grown with five solutions containing different proportions of nitrate and ammonium. Their nitrogen status was characterized through analyses of their biomass, different biochemical and molecular markers as well as a metabolite profile using 1H-NMR. Ammonium-fed seedlings exhibited higher biomass than nitrate-fed-seedlings. Nitrate mainly accumulated in the stem and ammonium in the roots. Needles of ammonium-fed seedlings had higher nitrogen and amino acid contents but lower levels of enzyme activities related to nitrogen metabolism. Higher amounts of soluble sugars and L-arginine were found in the roots of ammonium-fed seedlings. In contrast, L-asparagine accumulated in the roots of nitrate-fed seedlings. The differences in the allocation of nitrate and ammonium may function as metabolic buffers to prevent interference with the metabolism of photosynthetic organs. The metabolite profiles observed in the roots suggest problems with carbon and nitrogen assimilation in nitrate-supplied seedlings. Taken together, this new knowledge contributes not only to a better understanding of nitrogen metabolism but also to improving aspects of applied mineral nutrition for conifers.

NADH-GOGAT Overexpression Does Not Improve Maize (Zea mays L.) Performance Even When Pyramiding with NAD-IDH, GDH and GS.

Maize plants overexpressing NADH-GOGAT were produced in order to determine if boosting 2-Oxoglurate production used as a carbon skeleton for the biosynthesis of amino acids will improve plant biomass and kernel production. The NADH-GOGAT enzyme recycles glutamate and incorporates carbon skeletons into the ammonium assimilation pathway using the organic acid 2-Oxoglutarate as a substrate. Gene pyramiding was then conducted with NAD-IDH and NADH-GDH, two enzymes also involved in the synthesis of 2-Oxoglurate. NADH-GOGAT overexpression was detrimental for shoot biomass production but did not markedly affect kernel yield. Additional NAD-IDH and NADH-GDH activity did not improve plant performance. A decrease in kernel production was observed when NADH-GDH was pyramided to NADH-GOGAT and NAD-IDH. This decrease could not be restored even when additional cytosolic GS activity was present in the plants overexpressing the three enzymes producing 2-Oxoglutarate. Detailed leaf metabolic profiling of the different transgenic plants revealed that the NADH-GOGAT over-expressors were characterized by an accumulation of amino acids derived from glutamate and a decrease in the amount of carbohydrates further used to provide carbon skeletons for its synthesis. The study suggests that 2-Oxoglutarate synthesis is a key element acting at the interface of carbohydrate and amino acid metabolism and that its accumulation induces an imbalance of primary carbon and nitrogen metabolism that is detrimental for maize productivity.

Understanding plant nitrogen nutrition through a laboratory experiment.

Plant nitrogen nutrition is an essential topic in biology that should be included in scientific education. Nitrogen availability is one of the primary limiting factors for plant growth, and these organisms are the primary support for the pluricellular terrestrial life, since they are the fundamental group of autotrophs. For this reason, the use of nitrogen fertilizers is in the basis of the Green Revolution that led to an extraordinary increase in the food production during the twentieth century. To illustrate the importance of plant nitrogen nutrition, a new laboratory experience for students is presented in this manuscript. The aim of the following laboratory teaching activity is training of students through the evaluation of metabolic, biochemical, and molecular markers that are related to the nitrogen nutritional status of plants. For this purpose, cherry tomato plants (Solanum lycopersicum var. cerasiforme) were used, since they exhibit rapid growth and good differential biomass accumulation in response to changes in the nitrogen supply. The proposed laboratory experiment enables the development of the entire protocol for postgraduate students or in a reduced version for students from lower educational levels. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):450-458, 2019.

Nitrogen Metabolism and Biomass Production in Forest Trees.

Low nitrogen (N) availability is a major limiting factor for tree growth and development. N uptake, assimilation, storage and remobilization are key processes in the economy of this essential nutrient, and its efficient metabolic use largely determines vascular development, tree productivity and biomass production. Recently, advances have been made that improve our knowledge about the molecular regulation of acquisition, assimilation and internal recycling of N in forest trees. In poplar, a model tree widely used for molecular and functional studies, the biosynthesis of glutamine plays a central role in N metabolism, influencing multiple pathways both in primary and secondary metabolism. Moreover, the molecular regulation of glutamine biosynthesis is particularly relevant for accumulation of N reserves during dormancy and in N remobilization that takes place at the onset of the next growing season. The characterization of transgenic poplars overexpressing structural and regulatory genes involved in glutamine biosynthesis has provided insights into how glutamine metabolism may influence the N economy and biomass production in forest trees. Here, a general overview of this research topic is outlined, recent progress are analyzed and challenges for future research are discussed.

PpNAC1, a main regulator of phenylalanine biosynthesis and utilization in maritime pine.

The transcriptional regulation of phenylalanine metabolism is particularly important in conifers, long-lived species that use large amounts of carbon in wood. Here, we show that the Pinus pinaster transcription factor, PpNAC1, is a main regulator of phenylalanine biosynthesis and utilization. A phylogenetic analysis classified PpNAC1 in the NST proteins group and was selected for functional characterization. PpNAC1 is predominantly expressed in the secondary xylem and compression wood of adult trees. Silencing of PpNAC1 in P. pinaster results in the alteration of stem vascular radial patterning and the down-regulation of several genes associated with cell wall biogenesis and secondary metabolism. Furthermore, transactivation and EMSA analyses showed that PpNAC1 is able to activate its own expression and PpMyb4 promoter, while PpMyb4 is able to activate PpMyb8, a transcriptional regulator of phenylalanine and lignin biosynthesis in maritime pine. Together, these results suggest that PpNAC1 is a functional ortholog of the ArabidopsisSND1 and NST1 genes and support the idea that key regulators governing secondary cell wall formation could be conserved between gymnosperms and angiosperms. Understanding the molecular switches controlling wood formation is of paramount importance for fundamental tree biology and paves the way for applications in conifer biotechnology.

Analysis of the WUSCHEL-RELATED HOMEOBOX gene family in Pinus pinaster: New insights into the gene family evolution.

WUSCHEL-RELATED HOMEOBOX (WOX) genes are key players controlling stem cells in plants and can be divided into three clades according to the time of their appearance during plant evolution. Our knowledge of stem cell function in vascular plants other than angiosperms is limited, they separated from gymnosperms ca 300 million years ago and their patterning during embryogenesis differs significantly. For this reason, we have used the model gymnosperm Pinus pinaster to identify WOX genes and perform a thorough analysis of their gene expression patterns. Using transcriptomic data from a comprehensive range of tissues and stages of development we have shown three major outcomes: that the P. pinaster genome encodes at least fourteen members of the WOX family spanning all the major clades, that the genome of gymnosperms contains a WOX gene with no homologues in angiosperms representing a transitional stage between intermediate- and WUS-clade proteins, and that we can detect discrete WUS and WOX5 transcripts for the first time in a gymnosperm.

Characterization of Three L-Asparaginases from Maritime Pine (Pinus pinaster Ait.).

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.

The gene expression landscape of pine seedling tissues.

Conifers dominate vast regions of the Northern hemisphere. They are the main source of raw materials for timber industry as well as a wide range of biomaterials. Despite their inherent difficulties as experimental models for classical plant biology research, the technological advances in genomics research are enabling fundamental studies on these plants. The use of laser capture microdissection followed by transcriptomic analysis is a powerful tool for unravelling the molecular and functional organization of conifer tissues and specialized cells. In the present work, 14 different tissues from 1-month-old maritime pine (Pinus pinaster) seedlings have been isolated and their transcriptomes analysed. The results increased the sequence information and number of full-length transcripts from a previous reference transcriptome and added 39 841 new transcripts. In total, 2376 transcripts were ubiquitously expressed in all of the examined tissues. These transcripts could be considered the core 'housekeeping genes' in pine. The genes have been clustered in function to their expression profiles. This analysis reduced the number of profiles to 38, most of these defined by their expression in a unique tissue that is much higher than in the other tissues. The expression and localization data are accessible at ConGenIE.org (http://v22.popgenie.org/microdisection/). This study presents an overview of the gene expression distribution in different pine tissues, specifically highlighting the relationships between tissue gene expression and function. This transcriptome atlas is a valuable resource for functional genomics research in conifers.

Molecular fundamentals of nitrogen uptake and transport in trees.

Nitrogen (N) is frequently a limiting factor for tree growth and development. Because N availability is extremely low in forest soils, trees have evolved mechanisms to acquire and transport this essential nutrient along with biotic interactions to guarantee its strict economy. Here we review recent advances in the molecular basis of tree N nutrition. The molecular characteristics, regulation, and biological significance of membrane proteins involved in the uptake and transport of N are addressed. The regulation of N uptake and transport in mycorrhized roots and transcriptome-wide studies of N nutrition are also outlined. Finally, several areas of future research are suggested.

Exploiting the Genetic Diversity of Maize Using a Combined Metabolomic, Enzyme Activity Profiling, and Metabolic Modeling Approach to Link Leaf Physiology to Kernel Yield.

A combined metabolomic, biochemical, fluxomic, and metabolic modeling approach was developed using 19 genetically distant maize (Zea mays) lines from Europe and America. Considerable differences were detected between the lines when leaf metabolic profiles and activities of the main enzymes involved in primary metabolism were compared. During grain filling, the leaf metabolic composition appeared to be a reliable marker, allowing a classification matching the genetic diversity of the lines. During the same period, there was a significant correlation between the genetic distance of the lines and the activities of enzymes involved in carbon metabolism, notably glycolysis. Although large differences were observed in terms of leaf metabolic fluxes, these variations were not tightly linked to the genome structure of the lines. Both correlation studies and metabolic network analyses allowed the description of a maize ideotype with a high grain yield potential. Such an ideotype is characterized by low accumulation of soluble amino acids and carbohydrates in the leaves and high activity of enzymes involved in the C4 photosynthetic pathway and in the biosynthesis of amino acids derived from glutamate. Chlorogenates appear to be important markers that can be used to select for maize lines that produce larger kernels.

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.

Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.

BACKGROUND: In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution. RESULTS: In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82% of the gene structures, and a high proportion (85%) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members. CONCLUSIONS: The experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a Ruby gem for this class of analyses.

Poplar trees for phytoremediation of high levels of nitrate and applications in bioenergy.

The utilization of high amounts of nitrate fertilizers for crop yield leads to nitrate pollution of ground and surface waters. In this study, we report the assimilation and utilization of nitrate luxuriant levels, 20 times more than the highest N fertilizer application in Europe, by transgenic poplars overexpressing a cytosolic glutamine synthetase (GS1). In comparison with the wild-type controls, transgenic plants grown under high N levels exhibited increased biomass (171.6%) and accumulated higher levels of proteins, chlorophylls and total sugars such as glucose, fructose and sucrose. These plants also exhibited greater nitrogen-use efficiency particularly in young leaves, suggesting that they are able to translocate most of the resources to the above-ground part of the plant to produce biomass. The transgenic poplar transcriptome was greatly affected in response to N availability with 1237 genes differentially regulated in high N, while only 632 genes were differentially expressed in untransformed plants. Many of these genes are essential in the adaptation and response against N excess and include those involved in photosynthesis, cell wall formation and phenylpropanoid biosynthesis. Cellulose production in the transgenic plants was fivefold higher than in control plants, indicating that transgenic poplars represent a potential feedstock for applications in bioenergy. In conclusion, our results show that GS transgenic poplars can be used not only for improving growth and biomass production but also as an important resource for potential phytoremediation of nitrate pollution.

Transcriptome-wide analysis supports environmental adaptations of two Pinus pinaster populations from contrasting habitats.

BACKGROUND: Maritime pine (Pinus pinaster Aiton) grows in a range of different climates in the southwestern Mediterranean region and the existence of a variety of latitudinal ecotypes or provenances is well established. In this study, we have conducted a deep analysis of the transcriptome in needles from two P. pinaster provenances, Leiria (Portugal) and Tamrabta (Morocco), which were grown in northern Spain under the same conditions. RESULTS: An oligonucleotide microarray (PINARRAY3) and RNA-Seq were used for whole-transcriptome analyses, and we found that 90.95% of the data were concordant between the two platforms. Furthermore, the two methods identified very similar percentages of differentially expressed genes with values of 5.5% for PINARRAY3 and 5.7% for RNA-Seq. In total, 6,023 transcripts were shared and 88 differentially expressed genes overlapped in the two platforms. Among the differentially expressed genes, all transport related genes except aquaporins were expressed at higher levels in Tamrabta than in Leiria. In contrast, genes involved in secondary metabolism were expressed at higher levels in Tamrabta, and photosynthesis-related genes were expressed more highly in Leiria. The genes involved in light sensing in plants were well represented in the differentially expressed groups of genes. In addition, increased levels of hormones such as abscisic acid, gibberellins, jasmonic and salicylic acid were observed in Leiria. CONCLUSIONS: Both transcriptome platforms have proven to be useful resources, showing complementary and reliable results. The results presented here highlight the different abilities of the two maritime pine populations to sense environmental conditions and reveal one type of regulation that can be ascribed to different genetic and epigenetic backgrounds.