Biosynthesis of Phenylalanine in Plants

El aminoácido fenilalanina (Phe) desempeña en las plantas terrestres una función esencial, al actuar como precursor tanto de las proteínas como de la síntesis de fenilpropanoides, una amplia familia de metabolitos secundarios que cumplen funciones muy diversas y cuya aparición y diversificación está interrelacionada con la propia evolución de las plantas terrestres. La importancia de la síntesis de Phe es tal que se estima que más del 30% del CO2 fijado por las plantas en la fotosíntesis es finalmente derivado hacia la síntesis de este aminoácido, y de ahí hacia la biosíntesis de fenilpropanoides (Boerjan et ál., 2003), más particularmente ligninas, uno de los componentes fundamentales de las paredes celulares secundarias de las plantas. El objetivo general de esta tesis es obtener un mejor conocimiento la biosíntesis de fenilalanina en las plantas y su regulación en relación a la biosíntesis de metabolitos secundarios

The arogenate dehydratase ADT2 is essential for seed development in Arabidopsis

Phenylalanine (Phe) biosynthesis in plants is a key process, as Phe serves as precursor of proteins and phenylpropanoids. The prephenate pathway connects chorismate, final product of the shikimate pathway, with the biosynthesis of Phe and Tyr. Two alternative routes of Phe biosynthesis have been reported: one depending of arogenate, and the other of phenylpyruvate. Whereas the arogenate pathway is considered the main route, the role of the phenylpyruvate pathway remains unclear. Here, we report that the deficiency in ADT2, a bifunctional arogenate dehydratase (ADT)/ prephenate dehydratase (PDT) enzyme, causes embryo arrest and seed abortion. This result makes a clear distinction between the essential role of ADT2 and the five remaining ADTs from Arabidopsis, which display mostly overlapping functions. We have found that PHA2, a monofunctional PDT from yeast, restores the adt2 phenotype when is targeted within the plastids, but not when is expressed in the cytosol. Similar results can be obtained by expressing ADT3, a monofunctional ADT. These results suggest that Phe can be synthesized from phenylpyruvate or arogenate when the bifunctional ADT2 is replaced by other ADT or PDT enzymes during seed formation, highlighting the importance of Phe for embryo development, and providing further insights into the plasticity of Phe biosynthesis.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Biochemical regulation of arginine biosynthesis in plants

Arginine plays a relevant role in plant metabolism due to its importance as building block of proteins but also as precursor of multiple secondary metabolites, polyamines and nitric oxide. Importantly, arginine frequently plays an essential role as a major nitrogen storage form in seeds and other vegetative tissues and its mobilization provides an efficient flux of nitrogen for different physiological processes [1][2][3]. Despite its importance, the biochemical regulation and kinetics of the enzymes involved in arginine biosynthesis remains poorly characterized in plants. In this work, we provide new knowledge about the biochemical regulation of the three enzymes involved in the last steps of the arginine pathway: ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASSY), and argininosuccinate lyase (ASL). Our results indicate that these enzymes are regulated by the concentration of different amino acids and metabolites, including arginine, suggesting that feedback regulatory loops could play and important role in the homeostasis of this amino acid. Besides, these regulatory mechanisms seem to have been subjected to a progressive refinement during the evolution of land plants, pointing towards a coevolution with the higher requirements of arginine in seed plants.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Un dominio proteico de origen procariota sería esencial para una vía alternativa de biosíntesis de fenilalanina en plantas

En las plantas, la fenilalanina desempeña una importante función tanto como componente de las proteínas como precursor de la síntesis de fenilpropanoides, una amplia familia de metabolitos secundarios. La fenilalanina es sintetizada en los cloroplastos a partir de L-arogenato por la actividad arogenato deshidratasa (ADT). Recientemente se ha propuesto la existencia en plantas de una ruta alternativa de biosíntesis de Phe, que emplearía fenilpiruvato como intermediario, de manera similar a como ocurre en la gran mayoría de microorganismos. Esta ruta requeriría la enzima prefenato deshidratasa (PDT). Diferentes trabajos apuntan a que dicha actividad enzimática es realizada por las mismas proteínas que las ADT: de esta forma, se ha descrito que en Arabidopsis thaliana 2 de las 6 ADT son bifuncionales, presentando tanto actividad ADT como PDT in vitro. Mediante análisis filogenético y complementación funcional en levaduras, hemos identificado la existencia de al menos dos genes de Pinus pinaster codificantes de proteínas bifuncionales con actividad ADT/PDT. El análisis comparativo de estas secuencias, conjuntamente con experimentos de mutagénesis dirigida, ha permitido identificar un dominio de 22 aminoácidos en la región C-terminal que confiere actividad PDT a estas enzimas. Hemos denominado PAC (PDT Activity Conferring) a este dominio. Pueden encontrarse enzimas ADT con el dominio PAC en todos los linajes de plantas terrestres, además de en algas verdes, algas rojas y glaucofitas, los tres linajes evolutivos que surgieron de la adquisición de los cloroplastos. Estos resultados sugieren que la actividad PDT, y por tanto la capacidad de sintetizar Phe a través de fenilpiruvato, se ha conservado durante la evolución de las plantas y sus ancestros. La posibilidad de sintetizar Phe a través de dos rutas diferentes podría haber desempeñado una importante función en la evolución y regulación del metabolismo secundario en plantas.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Biosíntesis de fenilalanina en coníferas: la familia de las arogenato dehidratasas de Pinus pinaster

En las plantas, la biosíntesis de los aminoácidos aromáticos tirosina y fenilalanina tiene lugar en los cloroplastos a través de una ruta metabólica conocida como ruta del prefenato, y que tiene su origen en la ruta del siquimato. La biosíntesis de fenilalanina a través de la ruta del prefenato tiene lugar dos reacciones consecutivas: la primera de ellas consiste en la conversión de prefenato en arogenato a través de una reacción de transaminación (actividad prefenato-arogenato aminotransferasa, PAT) y posteriormente la transformación del arogenato en fenilalanina (actividad arogenato deshidratasa, ADT). Muy recientemente, dos nuevas publicaciones han aportado evidencias al respecto de la posibilidad de la existencia de una vía alternativa de biosíntesis de fenilalanina, que no dependería de arogenato (Yoo et al., 20013; de la Torre et al., 2014). En esta ruta alternativa el prefenato sería transformado en fenilpiruvato a través de la enzima prefenato deshidratasa (PDT). El fenilpiruvato producido en esta reacción sería posteriormente convertido en fenilalanina a través de una transaminasa de aminoácidos aromáticos. Una particularidad de especial interés es el hecho de que las actividades ADT y PDT se encuentran en las mismas proteínas en Arabidopsis thaliana (Cho et al., 2007). En los últimos años, nuestro grupo de investigación ha estado trabajando en la ruta del prefenato, centrados principalmente en la enzima prefenato-arogenato aminotransferasa de Pinus pinaster, y más recientemente en la caracterización de familia de las ADT en esta misma especie. La presente comunicación desarrolla el trabajo de caracterización que estamos realizando en la familia de genes ADT/PDT de P. pinaster, integrada por al menos 9 genes candidatos presentes en el transcriptoma de esta especie (Canales et al., 2013). Estos genes candidatos presentan patrones de expresión característicos y dependientes del órgano y el estadio de desarrollo de la planta. Adicionalmente, de estos 9 genes, 3 de ellos forman un grupo filogenético característico de gimnospermas. Los objetivos de nuestro trabajo se encuentran actualmente enfocados en la caracterización funcional de este grupo de 3 genes ADT/PDT característicos de coníferas. Canales J, Bautista R, Label P, Gómez-Maldonado J, Lesur I, Fernández-Pozo N, Rueda-López M, Guerrero-Fernández D, Castro-Rodríguez V, Benzekri H, Cañas RA, Guevara MA, Rodrigues A, Seoane P, Teyssier C, Morel A, Ehrenmann F, Le Provost G, Lalanne C, Noirot C, Klopp C, Reymond I, García- Gutiérrez A, Trontin JF, Lelu-Walter MA, Miguel C, Cervera MT, Cantón FR, Plomion C, Harvengt L, Avila C, Gonzalo Claros M, Cánovas FM. (2013). De novo assembly of maritime pine transcriptome: implications for forest breeding and biotechnology. Plant Biotechnol J. 12(3):286-99. Cho MH, Corea OR, Yang H, Bedgar DL, Laskar DD, Anterola AM, Moog-Anterola FA, Hood RL, Kohalmi SE, Bernards MA, Kang C, Davin LB and Lewis NG. (2007) Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases. J Biol Chem. 282(42):30827-35.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Phenylalanine biosynthesis: the role and evolution of arogenate dehydratase gene family in c

In plants, arogenate dehydratase activity (ADT, EC 4.2.1.91) is responsible for the last step in the main pathway for phenylalanine biosynthesis, known as the arogenate pathway, which consist in two steps: the conversion of prephenate to arogenate in a reaction catalyzed by the enzyme prephenate aminotransferase (PAT, EC 2.6.1.78) and the decarboxylation of arogenate to render phenylalanine catalyzed by ADT. The arogenate pathway results of particular interest according to the important role of phenylalanine in plant metabolism, acting as the main gate of entry to phenylpropanoids biosynthesis, that constitute up to 30 to 45% of plant organic matter (Razal et al., 1996). This is particularly relevant in perennial woody plants, in which lignification process and resultant biomass acumulation through plant life cycle are notably important. Despite of the high importance of phenylalanine biosynthesis and derived phenylpropanoids in plants biology, the arogenate pathway still remains poorly characterized, particularly in woody plants. Very recently, two independent publications reported physiological evidences suggesting an alternative arogenate-independent pathway for phenylalanine biosynthesis in plants (Yoo et al., 2013; De la Torre et al., 2014), as described previously in fungi and bacteria. This pathway is dependent of a prephenate dehydratase enzyme (PDT, EC 4.2.1.51) catalyzing the conversion of prephenate to phenylpyruvate, being subsequently converted into phenylalanine through a transamination reaction. It has been reported that ADT and PDT activities are housed in the same proteins in plants (Cho et al., 2007). Here we present preliminary results focused on the characterization of the ADT/PDT gene family in maritime pine (Pinus pinaster Ait.), a conifer tree of ecological and commercial interest. Our results demonstrate the existence of at least 9 ADT-like genes in the P. pinaster transcriptome, showing organ- and developmentspecific mRNA and protein expression profiles. Moreover, 3 of those 9 candidate genes present a distinctive phylogenetic clustering, forming a conifer-characteristic group of ADT-like genes differenced from the remaining ADT sequences. These findings highlights the potential importance of ADT/PDT activities in conifer metabolism, suggesting the existence of a singular and highly-specialized prephenate-related metabolism in conifers. Cho MH, Corea OR, Yang H, Bedgar DL, Laskar DD, Anterola AM, Moog-Anterola FA, Hood RL, Kohalmi SE, Bernards MA, Kang C, Davin LB and Lewis NG. (2007) Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases. J Biol Chem. 282(42):30827-35.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Biochemical identification and analysis of ADT-like proteins from plants

Phenylalanine (Phe) biosynthesis in plants is a fundamental process as Phe serves as a precursor of proteins but also of a massive array of essential phenylpropanoids for plant growth development defense or reproduction. Arogenate dehydratase (ADT) catalyzes the final and rate limiting step in the Phe biosynthetic pathway in plants the conversion of arogenate into Phe. In recent years different studies have shown that in most plant species multiple isogenes encode for alternative ADT isoenzymes that display partially overlapping roles in different aspects such as lignin accumulation or the size and mass of the stems (Corea et al. 2012a Corea et al. 2012b) but also display exclusive functions in processes related to development (El-Azaz et al. 2018) or accumulation of certain metabolites (Chen et al. 2016). Very interestingly in recent years it has been determined that some ADT isoenzymes also display prephenate dehydratase activity (PDT) and thus are able to participate in an alternative route of Phe synthesis in plants using phenylpyruvate as intermediary (El-Azaz et al. 2016). In our article El-Azaz et al. 2016 and using phylogenetic studies that included ADTs from multiple plant species corresponding to most plant clades we identified several sequences that encoded for proteins with partial similarity to plant ADTs that we have provisionally named as ADT-like proteins. Prominently in conifers we identified up to four ADT-like enzymes. Using functional complementation assays in yeast we have determined that these enzymes do not display PDT activity but remains unclear its putative ADT or alternative activity. Using biochemical structural and functional analysis we have investigated the role of these enzymes in the Pinus pinaster conifer species. This communication will discuss on how the neo-functionalization of these enzymes may be related to the existing metabolic specialization in some groups of plants.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A central role for bifunctional aspartate/prephenate aminotransferase in the biosynthesis of amino acids in plant plastids.

A central role for bifunctional aspartate/prephenate aminotransferase in the biosynthesis of amino acids in plant plastids. Fernando de la Torre, Jorge El-Azaz, Concepción Ávila, Francisco M. Cánovas Departamento de Biología Molecular y Bioquímica. Universidad de Málaga. Bifunctional aspartate/prephenate aminotransferases (AAT/PAT) are plastid-located enzymes encoded by a single locus in all reported plants, which develop two different enzymatic activities: aspartate aminotransferase (AAT), the reversible combination of L-Asp and α-ketoglutarate to render L-Glu and oxaloacetate, and prephenate aminotransferase (PAT), the reversible combination of L-Asp and prephenate to render oxaloacetate and arogenate (1), (2), (3). Interestingly, L-Asp and prephenate are direct precursors for the biosynthesis of six different amino acids within plant plastids: L-Phe, L-Tyr, L-Met, L-Thr, L-Ile and L-Lys. The genetic analysis of AAT/PAT-AT function in plants has remained elusive because its absence is lethal during embryogenesis as revealed by the study of Arabidopsis transposon mutants defective in female gametogenesis and embryo development (4). The observed lethal phenotype is consistent with the suppression of an essential enzyme for amino acid biosynthesis. Our attempts to obtain Arabidopsis transgenic plants suppressed for the corresponding At2g22250 gene were unsuccessful. In order to overcome this problem we chose to implement a different strategy using Virus Induced Gene Silencing in Nicotiana benthamiana, a useful technique to study the mature phenotype corresponding to the disruption of a gene described as essential during embryo development. N. benthamiana plants were successfully silenced for the endogenous NbAAT/PAT gene and exhibited severe reduction in growth, strong symptoms of chlorosis, and altered fresh weight-to-dry weight ratio in comparison with control plants (5). Metabolic analysis revealed that the silencing of NbAAT/PAT results in altered profiles of amino acids, chlorophylls, carbohydrates and very interestingly, affects lignin deposition in vascular bundles. Our results also demonstrates that both AAT and PAT activities, housed by NbAAT/PAT, are functional in plants and develops critical roles in the biosynthesis of amino acids in platids. Our biochemical and molecular data also provide consistent evidences with an alternative route for the biosynthesis of phenylalanine in plants similar to that described for various groups of microorganisms (5). Parallel research in petunia petals is also consistent with this hypothesis (6). (1)de la Torre et al. (2006) Plant Journal 46(3):414-425. (2)Maeda et al. (2011) Nat Chem Biol. 7(1):19-21. (3)de la Torre et al. (2009). Plant Physiology 149(4):1648-1660 (4)Pagnussat et al. (2005) Development 132:603-614. (5)de la Torre et al. (2014) Plant Physiology 164(1):92-104. (6)Yoo et al. (2013) Nat Commun. 25(4):2833.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Multidisciplinary teaching of Biotechnology and Omics sciences

In the last years, there was a great boom in the Omics fields that have developed as multidisciplinary sciences. They use laboratory techniques related to Biology and Chemistry but also Bioinformatics tools. However, the developmental progress of these disciplines has led that much of undergraduate studies related to Biology have curricula that become outdated. From this point of view, it is necessary to focus the students to the fundamentals and techniques of complementary disciplines that will be essentials for the understanding of the Omics sciences. In the present work, we have developed a new teaching approach for Biochemistry, Biology and Bioinformatics students. They formed interdisciplinary working groups. These groups have prepared and presented communications about different techniques or methods in Molecular Biology, Omics or Bioinformatics participating in a technical meeting. This learning strategy “I do and I learn” has enabled to the students a first contact with the scientific communication including the approach to the scientific literature to acquire technical knowledge. The cooperation between students from different disciplines has enriched their point of view and even has been used in some practical master’s works.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Maritime pine PpMYB8 directly co-regulates secondary cell wall architecture and the associated Phe-biosynthesis pathway

Plants rely on the biosynthesis of L-Phenylalanine as building block for the synthesis of proteins but also as precursor for a tremendous range of plant-derived compounds essential for its grown, development and defense. Polymerization of secondary cell wall in trees involves the massive biosynthesis, among others, of the Phe-derived compound lignin. Thus, these plants require an accurate metabolic coordination between Phe and lignin biosynthesis to ensure its normal development. We have here identified that the pine arogenate dehydratase, whose enzyme activity limits the biosynthesis of Phe in plants, is transcriptionally regulated through direct interaction with PpMyb8. We have also shown that this transcription factor is directly involve in secondary cell wall biogenesis and cell death processes. Together these results indicate that a single transcription factor coordinates lignin accumulation and the proper biosynthesis of its essential precursor L-Phe.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech