Rising to the Challenge: Developing Biosensors to Study Nitrogen Transport in Plants

Nitrogen (N) plays a crucial role in plant development and growth, which is why high concentrations of N in the form of fertilizers are commonly used in agricultural crops. However, the excess N present in fertilizers is predominantly leached into groundwater, resulting in contamination of one of the largest sources of drinking water and posing a significant threat to the environment and public health. It is therefore essential to comprehend the assimilation, transport, and biosynthesis of nitrogen compounds, in order to improve plant growth and development. Unfortunately, many of the biological and metabolic processes that occur at the cellular and subcellular levels in different organs, tissues, cells, and compartments in plants remain unknow due to the lack of tools available for real-time monitoring. The assimilation of inorganic and organic nitrogen is a complex process that involves numerous transporter systems present in plant cell membranes. These low/high affinity and low/high capacity transporters have been studied in different experimental plant models, but it is still unknown how they are distributed throughout the plant, as well as their modus operandi in each cellular type or intracellular compartment. In this study, we suggest creating and utilizing dual ratiometric biosensors equipped with fluorescent proteins in various subcellular compartments. Our proposal is based on previous findings on glutamate sensors in plants, which were tested in diverse cellular compartments (Castro-Rodriguez et al., 2021). To generate these biosensors, we identified promising candidates such as the ammonium transporter PpAMT1.3 (Castro-Rodriguez et al., 2016) and the amino acid permease PpAAP1 from a conifer plant model, Pinus pinaster (Llebres et al., 2022).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Pining for answers: Study of cationic amino acids (CATs) transport in maritime pine Pinus pinaster Aiton.

Amino acid (AA) transporters are membrane proteins involved in AA mobilization within and between cells and cells compartments. In plants, these transporters are responsible of AA transport from sink to source organelle/cell and vice versa. They have a variety of biological functions, including phloem loading and unloading, seed development, intracellular transport, and organic nitrogen (N) assimilation in roots (Dinkeloo et al., 2018). Widhalm et al. (2015) confirmed the existence of a cationic AA transporter in Petunia hybrida (PhCAT) that is involved in Phe transport from the plastids to the cytosol in flowers. Pinus pinaster is a conifer model tree with ecological and economical importance due to its forestry and biotechnological interest. Molecular studies have been developed, especially related with N transport, metabolism, and regulation as well as wood formation (Castro-Rodríguez et al., 2016, 2017; Ortigosa et al., 2020, 2022). The objective of this study is to identify cationic amino acid transporters (CATs) in maritime pine (P. pinaster) and to elucidate the intracellular and intercellular transport of amino acids. In our research group, 9 CATs have been identified in P. pinaster (PpCATs) using differential transcriptomic profiling in different tissues. The structure and topology of the membrane proteins, their gene expression levels and subcellular localization have been studied for PpCAT3, PpCAT9, PpCAT10 and PpCAT11. PpCAT3 and PpCAT9 are localized in the plasma membrane, PpCAT11 in the tonoplast while PpCAT10 is found in the chloroplast membrane. Furthermore, the localization of PpCAT10 was verified by chloroplast isolation and confirmed using an organelle marker. The location of PpCAT10 in the plastid membrane and its expression pattern suggest that this AA transporter has a critical role in AA transport from plastids to the cytosol (and/or vice versa) during seasonal growth in maritime pine.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Under pressure: Abies pinsapo Boiss, queen of the response to high temperature and water stress.

The Spanish fir (Abies pinsapo Boiss) is an ecologically and economically important conifer species, but little is known about its molecular responses to abiotic stresses related to climate change. The study of molecular responses in conifers faces challenges, including the lack of a complete genome and standardised protocols for handling of biological samples. Our group recently achieved the complete transcriptome assembly of A. pinsapo (Ortigosa et al. 2022), enabling functional genomic studies in this species. The objective of this work is to identify gene families related to the response to increased temperature and water stress in A. pinsapo. Initially, we focused our studies on LEA proteins (late embryogenesis abundant), dehydrins and HSPs (heat shock proteins) which are known to play important roles in stress responses in other species. We identified candidate genes and studied their differential expression patterns in various tissues of individuals from two geographical areas characterised by varying environmental conditions. Our results contribute to a better understanding of the molecular mechanisms that govern the response to abiotic stress in A. pinsapo and provide important information for the sustainability of natural and forest ecosystems in the south of the Iberian Peninsula which are vulnerable to the impact of climate change.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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 needsFunding for open access charge: Universidad de Malaga/CBUA

Emerging insights into nitrogen assimilation in gymnosperms

Gymnosperms are a heterogeneous and ancient group of seed plants that includes conifers, ginkgos, cycads and gnetophytes. Molecular studies on extant gymnosperms have been constrained by some discouraging features for experimental research such as their long life cycles, large sizes, complex megagenomes and abundant phenolic compounds in their woody tissues. However, the development of high-throughput sequencing and refined multiomics technologies in the last few years has allowed to explore the molecular basis of essential processes in this ancient lineage of plants. Nitrogen is one of the main limiting factors determining vascular development and biomass production in woody plants. Therefore, nitrogen uptake, metabolism, storage and recycling are essential processes for fundamental gymnosperm biology. Here, recent progress in the molecular regulation of nitrogen assimilation in gymnosperms is reviewed and some future perspectives on this topic are outlined.This research was fnancially supported by Ministry of Science and Innovation (BIO2015-73512-JIN, RTI2018-094041-B-I00 and PID2021-125040OB-I00) and by Junta de Andalucía (P20-00036 PAIDI 2020/FEDER, UE). JMVM was supported by a Grant from the Spanish Ministry of Education (FPU17/03517). Funding for open access publishing: Universidad Málaga/CBUA

Bajo estrés: búsqueda de genes relacionados con la respuesta al aumento de la temperatura y déficit hídrico en Abies pinsapo Boiss

El pinsapo (Abies pinsapo Boiss) es un árbol perteneciente a las coníferas, grupo que incluye especies de gran relevancia ecológica y económica. Actualmente, poco se conoce sobre su capacidad de resiliencia frente a los efectos del cambio climático, sin embargo, se sabe que las respuestas a nivel molecular ante un estrés ambiental involucran la expresión diferencial de numerosos genes en diferentes tipos celulares. El estudio de los mecanismos que rigen la adaptación y supervivencia de coníferas como el pinsapo se ha visto limitado por la falta de conocimientos básicos de sus genomas y por la baja disponibilidad de herramientas analíticas apropiadas. Recientemente, nuestro grupo ha conseguido el ensamblaje “de novo” del transcriptoma del pinsapo, proporcionando un amplio catálogo de genes (22.769) que se expresan en diferentes tejidos, de los cuales una elevada proporción (55%) son transcritos de longitud compleja (Ortigosa et al. 2022). Este recurso genómico ha permitido iniciar estudios de genómica funcional en esta especie para la caracterización de genes potencialmente implicados en la respuesta a estreses provocados por el cambio climático. En este trabajo se han realizado búsquedas de redes de genes involucrados en la respuesta al aumento de temperatura y el estrés hídrico. Entre los candidatos encontramos las proteínas LEA (late embryogenesis abundant) (Abdul et al. 2021), las deshidrinas (Sun et al. 2021) y las proteínas HSP (heat shock protein) (Jacob et al. 2017). Además, se han analizado los perfiles transcriptómicos de los genes de interés y se han establecido perfiles de expresión bajo diferentes condiciones ambientales. Esta información es de gran importancia para la sostenibilidad de ecosistemas naturales y forestales, y de relevancia ecológica para las montañas del sur de la península ibérica, vulnerables al impacto del cambio climático.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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Biotecnología del metabolismo del nitrógeno en plantas de interés forestal: implicaciones en bioeconomía

El concepto de bioeconomía tiene un papel cada vez más importante dentro del marco económico y ecológico global, considerándose hoy en día un pilar fundamental para un crecimiento sostenible. Un elemento esencial del desarrollo bioeconómico es la disponibilidad de una gran fuente renovable de biomasa y su conversión posteriormente en derivados de interés de diversa índole como materiales y energía. El Grupo de Biología Molecular y Biotecnología de la Universidad de Málaga desarrolla actividades encaminadas a conocer cómo el uso eficiente de los nutrientes nitrogenados determina el desarrollo vascular y la acumulación de biomasa en árboles. Los esfuerzos de investigación se han dirigido a estudiar la regulación molecular de la adquisición, asimilación y reciclaje de nitrógeno (N) para la biosíntesis de aminoácidos. Los estudios se han realizado en pino marítimo (Pinus pinaster Aiton), especie arbórea de gran importancia económica y ecológica en el área mediterránea y modelo relevante para la investigación genómica de coníferas. En el marco de varios proyectos europeos se han generado recursos genómicos y se ha establecido una plataforma tecnológica para la realización de estudios funcionales de genes en coníferas mediante embriogénesis somática. Se han producido líneas transgénicas (sobreexpresión y silenciamiento) para genes reguladores implicados en el metabolismo del N. Actualmente se exploran las propiedades moleculares y la regulación de genes implicados en la biosíntesis y el destino metabólico de la fenilalanina y la arginina, aminoácidos clave para la economía del N y la producción de biomasa en las coníferas. Se presenta una descripción general del programa de investigación y el papel de la biotecnología en el mismo.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech