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.

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.

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.