Involvement of Polyamines in Ribosome Biogenesis and Identification of Potential Genes Associated with Spermine Resistance in Arabidopsis thaliana

Abstract

[eng] Polyamines are small polycationic molecules that contain amino groups and are found in all living organisms. This study is aimed at acquiring a comprehensive understanding of the diverse roles of polyamines in plants, specifically in A. thaliana, in order to gain a better understanding of their mechanisms of action. Studies have revealed that polyamines increase general proteins synthesis and are necessary for efficient translation. While some progress has been made regarding the participation of polyamines in ribosome biogenesis in bacteria and, recently, also in mammalians, little is known about their contribution and mode of action in plants. Given our interest on plant defense, the first part of this study was focused on analyzing the ribosome complexes of plants treated with the polyamines putrescine (Put) and spermine (Spm) in combination or not with the purified pathogen associated molecular pattern (PAMP) flagellin22 (flg22). Our results evidenced that treatments with polyamines and flg22 lead to increases in polysome abundance, resulting in a global enhancement of translational activity. Riboproteomic analyses revealed the accumulation of specific ribosomal proteins in response to Put, but not to Spm. Put was also found in polysome fractions where Spm was absent, suggesting a specific effect for Put on ribosome function. Additionally, when analyzing mutants deficient in Put and Spm biosynthesis, we observed a differential accumulation of ribonucleoprotein complex binding proteins. The data suggests a new role for putrescine in plants and provide valuable information for future investigations on how polyamines may have modulatory and functional effects on the ribosome machinery. The second part of the study was focused on identifying mutants insensitive to Spm. For this, two strategies were followed: forward genetics through random EMS mutagenesis and reverse genetics through loss-of-function analyses. As part of our screening process, we administered exogenous Spm supplementation to identify mutants that display insensitivity to Spm. This is possible because exogenously supplied Spm inhibits the growth of A. thaliana seedlings in vitro, enabling us to pinpoint mutants that display resistance to Spm. The mutagenesis screen identified the Ammonium Transporter 2 (AMT2), the Putative Laccase 9 (LAC9), and a hypothetical protein (At5g28090) as candidate genes responsible for Spm tolerance in roots. Such tolerance was associated with reduced ROS production under Spm treatment. In addition, Copper-Amine Oxidase ɤ2 (CuAOɤ2) and mutations in ethylene pathway genes, EIN1, EIN2, EIN3, and EIN3EIL1, were identified as candidates underlying Spm tolerance in roots through reverse genetics. CuAOɤ2 mutation might be related to reduced ROS production. The lack of rescue using ethylene inhibitors and lack of reconstitution of the triple response by Spm feeding suggests an effect of Spm on ethylene signaling. Last, we conducted an RNA-seq experiment that revealed a correlation between exogenous Spm supplementation and responses to Fe deficiency. This correlation was further validated through RT-qPCR analysis, and the fact that Fe supplementation restored growth in photosynthetic tissues under Spm supplementation, along with the induction of coumarin production by exogenous Spm.

Additionally, we observed more severe Fe deficiency symptoms in spms mutants, indicating that Spm homeostasis can significantly impact the availability of Fe. The observed interaction between Spm and Fe deficiency holds promising implications in the modulation of the nutritional status of plants by polyamines. Overall, these studies open new frontiers of exploration and underscores the significance of polyamines in shaping various fundamental aspects of plant biology with potential applications in agriculture.

[spa] Las poliaminas son pequeñas moléculas policatiónicas formadas por varios grupos aminos y presentes en todos los organismos vivos. Este estudio tiene como objetivo obtener una comprensión completa de los diversos roles de las poliaminas en las plantas, específicamente en A. thaliana, con el fin de obtener una mejor comprensión de sus mecanismos de acción. La primera parte de este estudió se enfocó en analizar los complejos ribosomales de plantas tratadas con las poliaminas putrescina (Put) y espermina (Spm), en combinación o no con el patrón molecular asociado a patógenos (PAMP) flagelina22 (flg22). Nuestros resultados indican que la Put es importante en la maquinaria ribosomal de las plantas mientras que la Spm no es tan crucial. La segunda parte del estudio se centró en identificar mutantes insensibles a Spm utilizando dos estrategias. La aplicación de genética avanzada, a través de una mutagénesis aleatoria con EMS, nos permitió identificar al Transportador de Amonio 2 (AMT2), la Laccasa Putativa 9 (LAC9), y un gen que codifica una proteína hipotética (At5g28090) como candidatos responsables de la tolerancia a Spm en las raíces. Esta tolerancia se asoció con una reducción en la producción de especies reactivas de oxígeno (ROS) bajo el tratamiento de Spm. Además, el uso de genética inversa, mediante análisis de pérdida de función, nos permitió identificar la Cobre-Amino Oxidasa ɤ2 (CuAOɤ2) y genes de la vía de señalización del etileno, EIN1, EIN2, EIN3 e EIN3EIL1, como candidatos que subyacen a la tolerancia a Spm en las raíces. Nuestros resultados sugieren que CuAOɤ2 podría estar relacionado con la producción de (ROS) en presencia de Spm y que la presencia de Spm podría tener un efecto en la señalización del etileno. Además, nuestro estudio también revela una interacción entre la Spm y la deficiencia de Fe, con implicaciones prometedoras en la modulación del estado nutricional de las plantas mediante poliaminas. En resumen, estos estudios abren nuevas fronteras de exploración y destacan la importancia de las poliaminas en diversos aspectos fundamentales de la biología de las plantas, con aplicaciones potenciales en la agricultura.