Properties and Functional Analysis of Two Chorismate Mutases from Maritime Pine.

Through the shikimate pathway, a massive metabolic flux connects the central carbon metabolism with the synthesis of chorismate, the common precursor of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, as well as other compounds, including salicylate or folate. The alternative metabolic channeling of chorismate involves a key branch-point, finely regulated by aromatic amino acid levels. Chorismate mutase catalyzes the conversion of chorismate to prephenate, a precursor of phenylalanine and tyrosine and thus a vast repertoire of fundamental derived compounds, such as flavonoids or lignin. The regulation of this enzyme has been addressed in several plant species, but no study has included conifers or other gymnosperms, despite the importance of the phenolic metabolism for these plants in processes such as lignification and wood formation. Here, we show that maritime pine (Pinus pinaster Aiton) has two genes that encode for chorismate mutase, PpCM1 and PpCM2. Our investigations reveal that these genes encode plastidial isoenzymes displaying activities enhanced by tryptophan and repressed by phenylalanine and tyrosine. Using phylogenetic studies, we have provided new insights into the possible evolutionary origin of the cytosolic chorismate mutases in angiosperms involved in the synthesis of phenylalanine outside the plastid. Studies based on different platforms of gene expression and co-expression analysis have allowed us to propose that PpCM2 plays a central role in the phenylalanine synthesis pathway associated with lignification.

Under Stress: Searching for Genes Involved in the Response of Abies pinsapo Boiss to Climate Change.

Currently, Mediterranean forests are experiencing the deleterious effects of global warming, which mainly include increased temperatures and decreased precipitation in the region. Relict Abies pinsapo fir forests, endemic in the southern Iberian Peninsula, are especially sensitive to these recent environmental disturbances, and identifying the genes involved in the response of this endangered tree species to climate-driven stresses is of paramount importance for mitigating their effects. Genomic resources for A. pinsapo allow for the analysis of candidate genes reacting to warming and aridity in their natural habitats. Several members of the complex gene families encoding late embryogenesis abundant proteins (LEAs) and heat shock proteins (HSPs) have been found to exhibit differential expression patterns between wet and dry seasons when samples from distinct geographical locations and dissimilar exposures to the effects of climate change were analyzed. The observed changes were more perceptible in the roots of trees, particularly in declining forests distributed at lower altitudes in the more vulnerable mountains. These findings align with previous studies and lay the groundwork for further research on the molecular level. Molecular and genomic approaches offer valuable insights for mitigating climate stress and safeguarding this endangered conifer.

Monitoring nutrients in plants with genetically encoded sensors: achievements and perspectives.

Understanding mechanisms of nutrient allocation in organisms requires precise knowledge of the spatiotemporal dynamics of small molecules in vivo. Genetically encoded sensors are powerful tools for studying nutrient distribution and dynamics, as they enable minimally invasive monitoring of nutrient steady-state levels in situ. Numerous types of genetically encoded sensors for nutrients have been designed and applied in mammalian cells and fungi. However, to date, their application for visualizing changing nutrient levels in planta remains limited. Systematic sensor-based approaches could provide the quantitative, kinetic information on tissue-specific, cellular, and subcellular distributions and dynamics of nutrients in situ that is needed for the development of theoretical nutrient flux models that form the basis for future crop engineering. Here, we review various approaches that can be used to measure nutrients in planta with an overview over conventional techniques, as well as genetically encoded sensors currently available for nutrient monitoring, and discuss their strengths and limitations. We provide a list of currently available sensors and summarize approaches for their application at the level of cellular compartments and organelles. When used in combination with bioassays on intact organisms and precise, yet destructive analytical methods, the spatiotemporal resolution of sensors offers the prospect of a holistic understanding of nutrient flux in plants.

Identification of Metabolic Pathways Differentially Regulated in Somatic and Zygotic Embryos of Maritime Pine.

Embryogenesis is a complex phase of conifer development involving hundreds of genes, and a proper understanding of this process is critical not only to produce embryos with different applied purposes but also for comparative studies with angiosperms. A global view of transcriptome dynamics during pine somatic and zygotic embryogenesis is currently missing. Here, we present a genome-wide transcriptome analysis of somatic and zygotic embryos at three developmental stages to identify conserved biological processes and gene functions during late embryogenesis. Most of the differences became more significant as the developmental process progressed from early to cotyledonary stages, and a higher number of genes were differentially expressed in somatic than in zygotic embryos. Metabolic pathways substantially affected included those involved in amino acid biosynthesis and utilization, and this difference was already observable at early developmental stages. Overall, this effect was found to be independent of the line (genotype) used to produce the somatic embryos. Additionally, transcription factors differentially expressed in somatic versus zygotic embryos were analyzed. Some potential hub regulatory genes were identified that can provide clues as to what transcription factors are controlling the process and to how the observed differences between somatic and zygotic embryogenesis in conifers could be regulated.

Sponging of glutamate at the outer plasma membrane surface reveals roles for glutamate in development.

Plants use electrical and chemical signals for systemic communication. Herbivory, for instance, appears to trigger local apoplasmic glutamate accumulation, systemic electrical signals, and calcium waves that travel to report insect damage to neighboring leaves and initiate defense. To monitor extra- and intracellular glutamate concentrations in plants, we generated Arabidopsis lines expressing genetically encoded fluorescent glutamate sensors. In contrast to cytosolically localized sensors, extracellularly displayed variants inhibited plant growth and proper development. Phenotypic analyses of high-affinity display sensor lines revealed that root meristem development, particularly the quiescent center, number of lateral roots, vegetative growth, and floral architecture were impacted. Notably, the severity of the phenotypes was positively correlated with the affinity of the display sensors, intimating that their ability to sequester glutamate at the surface of the plasma membrane was responsible for the defects. Root growth defects were suppressed by supplementing culture media with low levels of glutamate. Together, the data indicate that sequestration of glutamate at the cell surface either disrupts the supply of glutamate to meristematic cells and/or impairs localized glutamatergic signaling important for developmental processes.

The amino acid permease PpAAP1 mediates arginine transport in maritime pine.

Forest trees have access to diverse nitrogenous compounds in the soil such as ammonium, nitrate and amino acids. Recent progress has been made in the identification and characterization of ammonium and nitrate transporters. However, much more limited is our current knowledge of amino acid transport systems despite their relevance to fully understanding nitrogen nutrition in trees. In the present study, we have identified 10 genes encoding putative amino acid permeases of the AAP family in maritime pine (Pinus pinaster Ait.). Four members of this family, PpAAP1, PpAAP2, PpAAP3 and PpAAP4 were phylogenetically related to AtAAP5, involved in arginine transport in Arabidopsis thaliana. One of these genes, PpAAP1, exhibited enhanced expression levels in maritime pine roots when arginine was externally supplied. PpAAP1 was functionally characterized by complementation of a yeast mutant strain defective in the transport of arginine, allowing yeast to take up [14C]-arginine with high affinity. Furthermore, PpAAP1 was able to restore the severely affected root uptake of arginine displayed by AtAAP5 T-DNA mutants. Uptake rates of 15N-labelled arginine were significantly higher in PpAAP1-overexpressing plants when compared to wild-type and AtAAP5 mutant plants. Taken together, our results indicate that PpAAP1 is a high-affinity arginine transporter in maritime pine.

Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses.

Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites in vivo. Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined in vitro such as substrate specificity, affinity, response range, dynamic range, and signal-to-noise ratio are important for evaluating in vivo data. This protocol provides a robust methodology for in vitro binding assays of newly designed sensors. Here we present a detailed protocol for purification and in vitro characterization of genetically encoded sensors, exemplified for the His affinity-tagged GO-(Green-Orange) MatryoshCaMP6s calcium sensor. GO-Matryoshka sensors are based on single-step insertion of a cassette containing two nested fluorescent proteins, circularly permutated fluorescent green FP (cpGFP) and Large Stoke Shift LSSmOrange, within the binding protein of interest, producing ratiometric sensors that exploit the analyte-triggered change in fluorescence of a cpGFP.

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.

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.

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.

Deciphering the molecular basis of ammonium uptake and transport in maritime pine.

Ammonium is the predominant form of inorganic nitrogen in the soil of coniferous forests. Despite the ecological and economic importance of conifers, the molecular basis of ammonium uptake and transport in this group of gymnosperms is largely unknown. In this study, we describe the functional characterization of members of the AMT gene family in Pinus pinaster: PpAMT1.1, PpAMT1.2 and PpAMT1.3 (subfamily 1) and PpAMT2.1 and PpAMT2.3 (subfamily 2). Our phylogenetic analysis indicates that in conifers, all members of the AMT1 subfamily evolved from a common ancestor that is evolutionarily related to the ancient PpAMT1.2 gene. Individual AMT genes are developmentally and nutritionally regulated, and their transcripts are specifically distributed in different organs. PpAMT1.3 was predominantly expressed in the roots, particularly during N starvation and mycorrhizal interaction, whereas PpAMT2.3 was preferentially expressed in lateral roots. Immunolocalization studies of roots with varied nitrogen availability revealed that PpAMT1 and PpAMT2 proteins play complementary roles in the uptake of external ammonium. Heterologous expression in yeast and Xenopus oocytes revealed that the AMT genes encode functional transporters with different kinetics and with different capacities for ammonium transport. Our results provide new insights on how nitrogen is acquired and transported in conifers.

Redundancy and metabolic function of the glutamine synthetase gene family in poplar.

BACKGROUND: Glutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is a key enzyme in ammonium assimilation and metabolism in higher plants. In poplar, the GS family is organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1.1, GS1.2 and GS1.3) and one group that codes for the choroplastic GS isoform (GS2). Our previous work suggested that GS duplicates may have been retained to increase the amount of enzyme in a particular cell type. RESULTS: The current study was conducted to test this hypothesis by developing a more comprehensive understanding of the molecular and biochemical characteristics of the poplar GS isoenzymes and by determinating their kinetic parameters. To obtain further insights into the function of the poplar GS genes, in situ hybridization and laser capture microdissections were conducted in different tissues, and the precise GS gene spatial expression patterns were determined in specific cell/tissue types of the leaves, stems and roots. The molecular and functional analysis of the poplar GS family and the precise localization of the corresponding mRNA in different cell types strongly suggest that the GS isoforms play non-redundant roles in poplar tree biology. Furthermore, our results support the proposal that a function of the duplicated genes in specific cell/tissue types is to increase the abundance of the enzymes. CONCLUSION: Taken together, our results reveal that there is no redundancy in the poplar GS family at the whole plant level but it exists in specific cell types where the two duplicated genes are expressed and their gene expression products have similar metabolic roles. Gene redundancy may contribute to the homeostasis of nitrogen metabolism in functions associated with changes in environmental conditions and developmental stages.

De novo assembly of maritime pine transcriptome: implications for forest breeding and biotechnology.

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.

The glutamine synthetase gene family in Populus.

BACKGROUND: Glutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is a key enzyme in ammonium assimilation and metabolism of higher plants. The current work was undertaken to develop a more comprehensive understanding of molecular and biochemical features of GS gene family in poplar, and to characterize the developmental regulation of GS expression in various tissues and at various times during the poplar perennial growth. RESULTS: The GS gene family consists of 8 different genes exhibiting all structural and regulatory elements consistent with their roles as functional genes. Our results indicate that the family members are organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1) and 1 which codes for the choroplastic GS isoform (GS2). Our analysis shows that Populus trichocarpa is the first plant species in which it was observed the complete GS family duplicated. Detailed expression analyses have revealed specific spatial and seasonal patterns of GS expression in poplar. These data provide insights into the metabolic function of GS isoforms in poplar and pave the way for future functional studies. CONCLUSIONS: Our data suggest that GS duplicates could have been retained in order to increase the amount of enzyme in a particular cell type. This possibility could contribute to the homeostasis of nitrogen metabolism in functions associated to changes in glutamine-derived metabolic products. The presence of duplicated GS genes in poplar could also contribute to diversification of the enzymatic properties for a particular GS isoform through the assembly of GS polypeptides into homo oligomeric and/or hetero oligomeric holoenzymes in specific cell types.