Biosíntesis, señalización y modo de acción del óxido nítrico en Arabidopsis thaliana

El óxido nítrico (NO) es una molécula gaseosa, muy reactiva, que ha ganado un creciente interés en los últimos años debido a su reconocido papel en señalización. Sin embargo, tanto su biosíntesis como su señalización o su modo de acción siguen siendo, en gran medida, desconocidas, lo que limita el avance del conocimiento de las bases moleculares de su función en plantas. En la presente tesis, hemos identificado las principales rutas enzimáticas de biosíntesis de NO en la planta modelo Arabidopsis thaliana mediante una aproximación genética. En un segundo paso, hemos utilizado los mutantes generados para establecer el papel del NO, y de cada una de sus rutas de biosíntesis, en diferentes procesos de desarrollo y de respuesta a estrés. Así, hemos descrito el papel negativo del NO en la sensibilidad al ácido abscísico en la regulación de la dormición y la germinación de las semillas, el establecimiento de plántula y el cierre estomático. Por otro lado, hemos definido el mecanismo molecular por el que el NO promueve la fotomorfogénesis. Brevemente, la producción de NO tras la transición de oscuridad a luz regula la expresión de genes clave de la fotomorfogénesis, como SLY1 y los factores de transcripción de tipo PIF, lo que promueve la des-etiolación que se concreta en una inhibición de la elongación del hipocotilo en condiciones de iluminación. Finalmente, para avanzar en el conocimiento sobre el modo de acción del NO, hemos caracterizado una de las modificaciones post-traduccionales mediadas por NO mediante el desarrollo de un método proteómico que nos permite enriquecer e identificar proteínas nitradas en residuos de tirosina. El trabajo desarrollado en esta tesis doctoral ha ayudado a avanzar en el conocimiento de los mecanismos moleculares por los que el NO regula algunos procesos fisiológicos en Arabidopsis thaliana y ha abierto numerosas puertas para el posterior análisis de muchos otros.Lozano Juste, J. (2011). Biosíntesis, señalización y modo de acción del óxido nítrico en Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11276Palanci

An Update on Crop ABA Receptors

[EN] The hormone abscisic acid (ABA) orchestrates the plant stress response and regulates sophisticated metabolic and physiological mechanisms essential for survival in a changing environment. Plant ABA receptors were described more than 10 years ago, and a considerable amount of information is available for the model plant Arabidopsis thaliana. Unfortunately, this knowledge is still very limited in crops that hold the key to feeding a growing population. In this review, we summarize genomic, genetic and structural data obtained in crop ABA receptors. We also provide an update on ABA perception in major food crops, highlighting specific and common features of crop ABA receptors.This research was funded by the grants SP20180340 (PAID-06-18), from Universitat Politecnica de Valencia, and GV/2020/159, from Generalitat Valenciana, to J.L.-J. and FONDOCYT2018 2C1-182 grant from FONDOCYT to S.M.R. and J.L.-J.Ruiz-Partida, R.; Rosario, SM.; Lozano Juste, J. (2021). An Update on Crop ABA Receptors. Plants. 10(6):1-22. https://doi.org/10.3390/plants10061087S12210

Unnatural agrochemical ligands for engineered abscisic acid receptors

[EN] Existing agrochemicals can be endowed with new applications through protein engineering of plant receptors. A recent study shows an engineered PYR1 ABA receptor can be activated by mandipropamid. Plants engineered with such PYR1 variant are responsive to this agrochemical, which confers protection against drought through activation of ABA signaling.Rodríguez Egea, PL.; Lozano Juste, J. (2015). Unnatural agrochemical ligands for engineered abscisic acid receptors. Trends in Plant Science. 20(6):330-332. doi:10.1016/j.tplants.2015.04.001S33033220

Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors

Nitric oxide (NO) is an important signaling compound in prokaryotes and eukaryotes. In plants, NO regulates critical developmental transitions and stress responses. Here, we identify a mechanism for NO sensing that coordinates responses throughout development based on targeted degradation of plant-specific transcriptional regulators, the group VII ethylene response factors (ERFs). We show that the N-end rule pathway of targeted proteolysis targets these proteins for destruction in the presence of NO, and we establish them as critical regulators of diverse NO-regulated processes, including seed germination, stomatal closure, and hypocotyl elongation. Furthermore, we define the molecular mechanism for NO control of germination and crosstalk with abscisic acid (ABA) signaling through ERF-regulated expression of ABSCISIC ACID INSENSITIVE5 (ABI5). Our work demonstrates how NO sensing is integrated across multiple physiological processes by direct modulation of transcription factor stability and identifies group VII ERFs as central hubs for the perception of gaseous signals in plants

Tribenuron-methyl metabolism and the rare Pro197Phe double mutation together with 2,4-D metabolism and reduced absorption can evolve in Papaver rhoeas with multiple and cross herbicide resistance to ALS inhibitors and auxin mimics

Multiple resistance mechanisms to ALS inhibitors and auxin mimics in two Papaver rhoeas populations were investigated in wheat fields from Portugal. Dose-response trials, also with malathion (a cytochrome P450 inhibitor), cross-resistance patterns for ALS inhibitors and auxin mimics, alternative herbicides tests, 2,4-D and tribenuron-methyl absorption, translocation and metabolism experiments, together with ALS activity, gene sequencing and enzyme modelling and ligand docking were carried out. Results revealed two different resistant profiles: one population (R1) multiple resistant to tribenuron-methyl and 2,4-D, the second (R2) only resistant to 2,4-D. In R1, several target-site mutations in Pro197 and enhanced metabolism (cytochrome P450-mediated) were responsible of tribenuron-methyl resistance. For 2,4-D, reduced transport was observed in both populations, while cytochrome P450-mediated metabolism was also present in R1 population. Moreover, this is the first P. rhoeas population with enhanced tribenuron-methyl metabolism. This study reports the first case for P. rhoeas of the amino acid substitution Pro197Phe due to a double nucleotide change. This double mutation could cause reduced enzyme sensitivity to most ALS inhibitors according to protein modelling and ligand docking. In addition, this study reports a P. rhoeas population resistant to 2,4-D, apparently, with reduced transport as the sole resistance mechanism.This work has been supported by the Asociacion de Agroquimicos y Medio Ambiente, Spain. Joel Torra acknowledges support from the Spanish Ministry of Science, Innovation, and Universities (grant Ramon y Cajal RYC2018–023866-I) and by the Spanish State Research Agency, Spain (AEI) and the European Regional Development Fund, EU (ERDF) through the projects AGL2017-83325-C4-2-R and PID2020-113229RB- C42. The field surveys made in Portugal were supported by Foundation for Science and Technology through the project UIDB/05064/2020 (VALORIZA). Jorge Lozano-Juste group is funded by grants RYC2020- 029097-I and PID2021-128826OA-I00 from Ministerio de Ciencia e Innovaci ́on (MCIN, Spain), AEI and the ERDF

Pre-mRNA splicing repression triggers abiotic stress signaling in plants

[EN] Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress-and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants.We wish to thank members of the Laboratory for Genome Engineering at King Abdullah University of Science and Technology for helpful discussions and comments on the manuscript. We wish to thank Moussa Benhamed for helpful discussions and suggestions and for providing key materials. We wish to thank Sean Cutler for providing Arabidopsis seeds of MAKPKKK18-uidA. This study was supported by King Abdullah University of Science and Technology. Work in PR's laboratory was funded by grant BIO2014-52537-R from MINECO. Work in PD's laboratory is funded by grant PTDC/BIA-PLA/1084/2014 from FCT. The authors declare no conflicts of interest.Ling, Y.; Alshareef, S.; Butt, H.; Lozano Juste, J.; Li, L.; Galal, AA.; Moustafa, A.... (2017). Pre-mRNA splicing repression triggers abiotic stress signaling in plants. The Plant Journal. 89(2):291-309. https://doi.org/10.1111/tpj.13383S29130989

Abscisic acid mimic-fluorine derivative 4 alleviates water deficit stress by regulating ABA-responsive genes, proline accumulation, CO2 assimilation, water use efficiency and better nutrient uptake in tomato plants

Water deficit represents a serious limitation for agriculture and both genetic and chemical approaches are being used to cope with this stress and maintain plant yield. Next-generation agrochemicals that control stomatal aperture are promising for controlling water use efficiency. For example, chemical control of abscisic acid (ABA) signaling through ABA-receptor agonists is a powerful method to activate plant adaptation to water deficit. Such agonists are molecules able to bind and activate ABA receptors and, although their development has experienced significant advances in the last decade, few translational studies have been performed in crops. Here, we describe protection by the ABA mimic-fluorine derivative 4 (AMF4) agonist of the vegetative growth in tomato plants subjected to water restriction. Photosynthesis in mock-treated plants is markedly impaired under water deficit conditions, whereas AMF4 treatment notably improves CO2 assimilation, the relative plant water content and growth. As expected for an antitranspirant molecule, AMF4 treatment diminishes stomatal conductance and transpiration in the first phase of the experiment; however, when photosynthesis declines in mock-treated plants as stress persists, higher photosynthetic and transpiration parameters are recorded in agonist-treated plants. Additionally, AMF4 increases proline levels over those achieved in mock-treated plants in response to water deficit. Thus water deficit and AMF4 cooperate to upregulate P5CS1 through both ABA-independent and ABA-dependent pathways, and therefore, higher proline levels are produced Finally, analysis of macronutrients reveals higher levels of Ca, K and Mg in AMF4- compared to mock-treated plants subjected to water deficit. Overall, these physiological analyses reveal a protective effect of AMF4 over photosynthesis under water deficit and enhanced water use efficiency after agonist treatment. In summary, AMF4 treatment is a promising approach for farmers to protect the vegetative growth of tomatoes under water deficit stress

Structure-guided engineering of a receptor-agonist pair for inducible activation of the ABA adaptive response to drought

Strategies to activate abscisic acid (ABA) receptors and boost ABA signaling by small molecules that act as ABA receptor agonists are promising biotechnological tools to enhance plant drought tolerance. Protein structures of crop ABA receptors might require modifications to improve recognition of chemical ligands, which in turn can be optimized by structural information. Through structure-based targeted design, we have combined chemical and genetic approaches to generate an ABA receptor agonist molecule (iSB09) and engineer a CsPYL1 ABA receptor, named CsPYL15m, which efficiently binds iSB09. This optimized receptor-agonist pair leads to activation of ABA signaling and marked drought tolerance. No constitutive activation of ABA signaling and hence growth penalty was observed in transformed Arabidopsis thaliana plants. Therefore, conditional and efficient activation of ABA signaling was achieved through a chemical-genetic orthogonal approach based on iterative cycles of ligand and receptor optimization driven by the structure of ternary receptor-ligand-phosphatase complexes

Involvement of nitric oxide (NO) and auxin in signal transduction of copper induced morphological responses in Arabidopsis seedlings

Background and Aims Plants are able to adapt to the environment dynamically through regulation of their growth and development. Excess copper (Cu2+ ), a toxic heavy metal, induces morphological alterations in plant organs; however, the underlying mechanisms are still unclear. With this in mind, the multiple signalling functions of nitric oxide (NO) in plant cells and its possible regulatory role and relationship with auxin were examined during Cu2+ -induced morphological responses. Methods Endogenous auxin distribution was determined by microscopic observation of X-Gluc-stained DR5::GUS arabidopsis, and the levels of NO, superoxide and peroxynitrite were detected by ¿uorescence microscopy. As well as wild-type, NO-overproducer (nox1) and -de¿cient (nia1nia2 and nia1nia2noa1-2) arabidopsis plants were used. Key Results Cu2+ at a concentration of 50mM resulted in a large reduction in cotyledon area and hypocotyl and primary root lengths, accompanied by an increase in auxin levels. In cotyledons, a low Cu2+ concentration promoted NO accumulation, which was arrested by nitric oxide synthase or nitrate reductase inhibitors. The 5-mM Cu2+ -induced NO synthesis was not detectable in nia1nia2 or nia1nia2noa1-2 plants. In roots, Cu2+ caused a decrease of the NO level which was not associated with superoxide and peroxynitrite formation. Inhibition of auxin transport resulted in an increase in NO levels, while exogenous application of an NO donor reduced DR5::GUS expression. The elongation processes of nox1 were not sensitive to Cu2+ , but NO-de¿cient plants showed diverse growth responses. ConclusionsIn plant organs, Cu2+ excess results in severe morphological responses during which the endogenous hormonal balance and signal transduction are affected. Auxin and NO negatively regulate each other¿s level and NO intensi¿es the metal-induced cotyledon expansion, but mitigates elongation processes under Cu2+ exposurePetó, A.; Lehotai, N.; Lozano Juste, J.; Leon Ramos, J.; Tari, I.; Erdei, L.; Kolbert, Z. (2011). Involvement of nitric oxide (NO) and auxin in signal transduction of copper induced morphological responses in Arabidopsis seedlings. Annals of Botany. 108(3):449-457. doi:10.1093/aob/mcr176S449457108

Pre-mRNA splicing repression triggers abiotic stress signaling in plants

Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work showed that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various AS inhibitors. Here, we show that the macrolide Pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA-seq data revealed that PB mimics abiotic stress signals including salt, drought, and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A::LUC and MAPKKK18::GUS in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2Cs phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling, and post-transcriptional regulation in plants.</p