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15 Pags.- 11 Figs. © The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License., The ornithine–urea cycle (urea cycle) makes a significant contribution to the metabolic responses of lower photosynthetic eukaryotes to episodes of high nitrogen availability. In this study, we compared the role of the plant urea cycle and its relationships to polyamine metabolism in ammonium-fed and nitrate-fed Medicago truncatula plants. High ammonium resulted in the accumulation of ammonium and pathway intermediates, particularly glutamine, arginine, ornithine, and putrescine. Arginine decarboxylase activity was decreased in roots, suggesting that the ornithine decarboxylase-dependent production of putrescine was important in situations of ammonium stress. The activity of copper amine oxidase, which releases ammonium from putrescine, was significantly decreased in both shoots and roots. In addition, physiological concentrations of ammonium inhibited copper amine oxidase activity in in vitro assays, supporting the conclusion that high ammonium accumulation favors putrescine synthesis. Moreover, early supplementation of plants with putrescine avoided ammonium toxicity. The levels of transcripts encoding urea-cycle-related proteins were increased and transcripts involved in polyamine catabolism were decreased under high ammonium concentrations. We conclude that the urea cycle and associated polyamine metabolism function as important protective mechanisms limiting ammonium toxicity in M. truncatula. These findings demonstrate the relevance of the urea cycle to polyamine metabolism in higher plants., This work was supported by the grants from the Spanish Government AGL2014-52396-P (MICINN) and AGL2017-86293-P (MINECO/FEDER) to JFM, and the Basque Government, Spain, IT-1018-16 (UPV/EHU-GV) to RE. MU is a recipient of a pre-doctoral fellowship from the Government of Navarre, Spain. JB and PLG have received pre-doctoral fellowships from the Public University of Navarre, Spain. PT has received funding from the Italian Ministry of Education, University and Research (Grant to Department of Science, University ‘Roma Tre’-‘Dipartimenti di Eccellenza’, ARTICOLO 1, COMMI 314–337. LEGGE 423 232/2016; PRIN 2017—CUP F84I19000730005). Partial support was obtained from the Spanish State Research Agency AGL2017-83358-R (AEI/FEDER) and the Government of Aragón, Spain, Group A09-20R to YG. Open Access funding was provided by the Public University of Navarra.

Iron (Fe) is an essential plant micronutrient, being a major limiting growth factor in calcareous soils. To increase Fe uptake, plants induce lateral roots growth, the expression of a Fe(III)-chelate reductase (FCR), a Fe(II)-transporter and a H+-ATPase and the secretion of flavins. Furthermore, auxin hormone family is involved in the Fe-deficiency responses but the action mechanism remains elusive. In this work, we evaluated the effect of the auxin-precursor indole-3-acetaldoxime (IAOx) on hydroponically grown Medicago truncatula plants under different Fe conditions. Upon 4-days of Fe starvation, the pH of the nutrient solution decreased, while both the FCR activity and the presence of flavins increased. Exogenous IAOx increased lateral roots growth contributing to superroot phenotype, decreased chlorosis, and delayed up to 3-days the pH-decrease, the FCR-activity increase, and the presence of flavins, compared to Fe-deficient plants. Gene expression levels were in concordance with the physiological responses. Results: showed that IAOx was immediately transformed to IAN in roots and shoots to maintain auxin homeostasis. IAOx plays an active role in iron homeostasis delaying symptoms and responses in Fe-deficient plants. We may speculate that IAOx or its derivatives remobilize Fe from root cells to alleviate Fe-deficiency. Overall, these results point out that the IAOx-derived phenotype may have advantages to overcome nutritional stresses. This work was supported by the Spanish Research Agency [grant AGL2017-83358-R funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”] and the Government of Aragón [grant A09_23R], co-financed with FEDER funds; and the CSIC [grant 2020AEP119] to Y.G. This research was partially supported by [grants AGL2017-52396-P funded from MINECO (co-financed with FEDER funds) and PID-2020-1177 03GB-I00 from the Public University of Navarre] to J.F.M. J.M., M.U. and J.B. were the recipients of pre-doctoral contracts awarded by the Government of Aragon, Government of Navarre, and Public University of Navarre, Spain, respectively. A.R. was hired [grants AGL2017-83358-R and 2020AEP119]. We acknowledge support of the publication fee by the CSIC Gold Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

El óxido nítrico (NO) es una molécula señalizadora ampliamente conocida en el mundo vegetal, relacionada con cada etapa en el desarrollo de la planta. De entre todas sus funciones descritas, se sabe que actúa sinérgicamente con el ácido indol-3-acético (IAA), promoviendo el desarrollo de raíces secundarias. Hasta ahora tan solo se han confirmado algunas vías de síntesis del NO, todas ellas reductivas, mientras que aún no se ha revelado vía oxidativa alguna. Varios informes de nuestro grupo de investigación han medido síntesis de novo de NO3- y NO2- en Pisum sativum y M. truncatula crecidas con NH4+ como única fuente de nitrógeno (datos sin publicar). Este hecho sugiere la existencia de una vía oxidativa para el NH4+ en fabáceas. Se propone también que este mismo mecanismo puede ser parte de la señalización por toxicidad de NH4+ y de los procesos para su mitigación. Dada su configuración molecular, las oximas son buenos candidatos para ser precursores del NO, y, por tanto, el primer paso de esta vía de oxidación de nitrógeno. Entre todas las oximas, la Indol-3-acetaldoxima (IAOx) es especialmente relevante ya que se sitúa en la encrucijada entre el IAA y los indol glucosinolatos. El papel del IAOx en el desarrollo y señalización está muy poco estudiado en crucíferas, y es prácticamente desconocido en otras familias. En esta tesis doctoral intentamos demostrar que el IAOx está presente en M. truncatula y que posee, además, importantes funciones de señalización durante el desarrollo radicular. Por último, hipotetizamos que la señalización de IAOx está mediada por NO. Para esta labor, hemos sintetizado IAOx y una colección de otras oximas indólicas y no indólicas puras y hemos utilizado un enfoque farmacológico utilizando la planta leguminosa modelo M. truncatula. Hemos medido el fenotipo radicular, cuantificado los compuestos indólicos en tejido (parte aérea y raíz) y medido la expresión de los genes de Indol-3-acetaldehido oxidasa e IAOx deshidratasa. Nuestros datos muestran que todas las oximas promueven el fenotipo ‘superoot’, concordando con nuestra hipótesis de que el IAOx produce su efecto a través de la liberación de NO. Este nuevo conocimiento es un gran paso hacia el descubrimiento de la vía oxidativa de síntesis de NO en plantas y arroja luz a la interacción entre IAOx, IAA y la nutrición nitrogenada, que será imprescindible para futuras investigaciones en campos de cultivo Nitric oxide (NO) is a widely recognized signalling molecule in plants. It affects almost every developmental step during the whole plant’s lifespan. Among all of its already described functions, NO is recognised to act synergistically with Indole-3-acetic acid (IAA), promoting the development of the secondary roots. Until now, only a few reductive NO synthesis pathways have been confirmed, whereas no oxidative pathway has been yet described. Experiments of our research group measured de novo synthesis of NO3- and NO2- in Pisum sativum and M. truncatula grown with NH4+ as the sole N source (unpublished data). This fact suggests the existence of an oxidative pathway for NH4+ in the Fabaceae family, which is proposed to be part of the signalling of the NH4+ toxicity and to participate in the alleviation mechanism. Due to their molecular configuration, oximes are very strong candidates for being the precursors of NO, and thus the first step into this nitrogen oxidation pathway. Among these oximes, Indole-3-acetaldoxime (IAOx) is particularly relevant since it is placed in the crossroad between IAA and indole glucosinolates. The role of IAOx in growth-signalling and root phenotype is poorly studied in cruciferous plants and mostly unknown in non-cruciferous plants. In this PhD thesis, we aim to demonstrate that IAOx is present in M. truncatula playing an important role of signalling during plant root development and also that this signalling is mediated by NO. For that purpose, we synthesized a set of pure IAOx and other indolic and non-indolic oximes and performed pharmacological approaches with the model legume M. truncatula. We analysed the root phenotype, quantified the indolic compounds in tissue (shoots and roots) and measured the Indole-3- acetaldehyde oxidase and IAOx dehydratase genes expression. Our data showed that all the oximes promoted the ‘superoot’ phenotype. All this matches with the hypothesis that IAOx exerts its signalling by liberating NO. This new knowledge is a step forward towards the discovery of an oxidative NO synthesis pathway in plants and throws light into the interplay between IAOx, IAA and nitrogen nutrition, which will be paramount for further field research in crop production. Esta tesis se ha realizado en el marco de los proyectos AGL2014-52396-P del Ministerio de Economía y Competitividad del Gobierno de españa y UPV/EHU-GV IT-1018-16 del Gobierno Vasco. Ayuda predoctoral de formacion de nuevo personal investigador de la UPNA y una ayuda predoctoral de movilidad internacional. Programa de Doctorado en Agrobiología Ambiental (RD 99/2011) Ingurumen Agrobiologiako Doktoretza Programa (ED 99/2011)

Plants differ widely in their growth and tolerance responses to ammonium and urea nutrition, while derived phenotypes seem markedly different from plants grown under nitrate supply. Plant responses to N sources are complex, and the traits involved remain unknown. This work reports a comprehensive and quantitative root proteomic study on the NH4+-tolerant legume Medicago truncatula grown under axenic conditions with either nitrate, NH4+ or urea supply as sole N source by using the iTRAQ method. Sixty-one different proteins among the three N sources were identified. Interestingly, among the proteomic responses, urea nutrition displayed greater similarity to nitrate than to ammonium nutrition. We found remarkable differences in membrane proteins that play roles in sensing the N form, and regulate the intracellular pH and the uptake of N. Also, several groups of proteins were differentially expressed in the C metabolism pathway involved in reorganizing N assimilation. In addition, enzymes related to phenylpropanoid metabolism, including the peroxidases POD2, POD6, POD7 and POD11, which were up-regulated under ammonium nutrition, contributed to the reinforcement of cell walls, as confirmed by specific staining of lignin. Thus, we identified cell wall lignification as an important tolerance mechanism of root cells associated with the stunted phenotype typical of plants grown under ammonium nutrition. This work was supported by the grants AGL2014-52396-P and AGL2017-86293-P from the Spanish Ministry of Economy and Competitiveness-MINECO. RE received a Juan de la Cierva-incorporación grant IJCI-2014-21452. JB is a holder of a PhD fellowship from the Public University of Navarre. The Proteomics Unit of Navarrabiomed is a member of ProteoRed, PRB3-ISCIII-Spain, and is supported by grant PT17/0019/009, of the PE I + D+I 2018-2020 funded by ISCIII and FEDER.

Soybean is a crop of agronomic importance that requires adequate watering during its growth to achieve high production. In this study, we determined physiological, photochemical and metabolic differences in five soybean varieties selected from the parental lines of a nested association mapping population during mild drought. These varieties have been described as high yielding (NE3001, HY1; LD01‐5907, HY2) or drought tolerant (PI518751; HYD1; PI398881, HYD2). Nevertheless, there has been little research on the physiological traits that sustain their high productivity under water‐limited conditions. The results indicate that high‐yielding varieties under drought cope with the shortage of water by enhancing their photoprotective defences and invest in growth and productivity, linked to a higher intrinsic water use efficiency. This is the case of the variety N‐3001 (HY1), with a tolerance strategy involving a faster transition into the reproductive stage to avoid the drought period. The present study highlights the role of the physiological and biochemical adjustments of various soybean varieties to cope with water‐limited conditions. Moreover, the obtained results underscore the fact that the high phenotypic plasticity among soybean phenotypes should be exploited to compensate for the low genetic variability of this species when selecting plant productivity in constrained environments., This work was supported by grants from the Spanish Ministry of Science and Innovation and FEDER funds (AGL2014‐52396) and from the Basque Government (UPV/EHU‐GV IT‐1018‐16; IT‐932‐16). J.B. is a holder of a PhD fellowship from the Public University of Navarre. R.E. received a Juan de la Cierva‐incorporación grant (IJCI‐2014‐21452).

The consumption of zeaxanthin (Z) through a vegetable-rich diet is recommended to reduce the progression of age-related macular degeneration. Due to Z’s intrinsic dynamic character that results from its participation in the photoprotective xanthophyll cycle involving the carotenoids violaxanthin, antheraxanthin and zeaxanthin (VAZ), post-harvest handling practices and storage usually retain low amounts of this bioactive compound (compared to the rest of phytochemicals that are, in general, more stable). Thus, the aim of this work was to investigate in important consumed leafy vegetables the effects of different storage conditions on carotenoids (mainly Z) including i) packaging under three modified atmospheres (MAs), ii) light refrigerated supermarket storage and iii) dark refrigerated domestic storage. The results showed that an MA with low O2 and high CO2 enhanced the Z content under light. Moreover, both light and dark refrigerated storage showed dynamic and circadian pigment changes that enhanced the total VAZ pool. These results can contribute to generating practical recommendations for industries, supermarkets, and consumers when high Z content is a nutritional target. RE received a Juan de la Cierva-incorporación grant IJCI-2014-21452. JB is a holder of a PhD fellowship from the Public University of Navarre. This research was supported by research BFU 2010-15021 and CTM2014- 53902-C2-2-P from the Ministry of Education and Science of Spain and the ERDF (FEDER) and research project UPV/EHU IT-1018-16. Technical and human support by Eroski supermarkets and “Fundación Tecnova” is gratefully acknowledged.

Indole-3-acetaldoxime (IAOx) is a particularly relevant molecule as an intermediate in the pathway for tryptophan-dependent auxin biosynthesis. The role of IAOx in growth-signalling and root phenotype is poorly studied in cruciferous plants and mostly unknown in non-cruciferous plants. We synthesized IAOx and applied it to M. truncatula plants grown axenically with NO3 -, NH4 + or urea as the sole nitrogen source. During 14 days of growth, we demonstrated that IAOx induced an increase in the number of lateral roots, especially under NH4 + nutrition, while elongation of the main root was inhibited. This phenotype is similar to the phenotype known as superroot previously described in SUR1- and SUR2-defective Arabidopsis mutants. The effect of IAOx, IAA or the combination of both on the root phenotype was different and dependent on the type of N-nutrition. Our results also showed the endogenous importance of IAOx in a legume plant in relation to IAA metabolism, and suggested IAOx long-distance transport depending on the nitrogen source provided. Finally, our results point out to CYP71A as the major responsible enzymes for IAA synthesis from IAOx, while they exclude indole-3-acetaldehyde oxidases. © 2019 Elsevier B.V. This work was supported by the grants AGL2017-86293-P and CGL2017-84723-P from the Spanish Ministry of Economy and Competitiveness (MINECO) , AGL2014-52396-P from the Ministry of Science Innovation and Universities (MICINN) , and IT932-16 from the Basque Government, Spain . JB and PL-G are holders of PhD fellowships from the Public University of Navarre. ACh received a Juan de la Cierva initiation grant FJCI-2016-27905 and RE received a Juan de la Cierva incorporation grant IJCI-2014-21452. This research was also supported by the Basque Government through the BERC 2018-2021 program, and by the Spanish Ministry of Science, Innovation and Universities through the BC3 María de Maeztu excellence accreditation (MDM-2017-0714).

Humans are continuously exposed to oxidative damage risk and in order to counteract it, the consumption of antioxidants and carotenoids of plant origin is recommended. Numerous studies show the need to include substantial amounts of the carotenoid zeaxanthin (Z) in the diet, because its deficiency provokes the development of macular degeneration, which leads to irreversible loss of vision. However, Z is a less-abundant carotenoid in plants, because most of its pool is rapidly converted to the carotenoid violaxanthin (V) via antheraxanthin (A), due to its involvement in the operation of the xanthophyll (V. +. A. +. Z) cycle. The aim of this paper, therefore, was to develop a protocol to enhance the Z content in spinach and rocket through two strategies: firstly, by applying stress (chilling, high light and drought) in order to enhance the total pool of V. +. A. +. Z and, secondly, by applying post-harvest treatments before consumption in order to enhance Z formation. The results showed that high light was the most beneficial stress, increasing the fresh weight production in rocket and showing the highest accumulation of V. +. A. +. Z and carotenoids in both species. An enhancement of α-tocopherol in rocket was, as well, accomplished by the environmental stress induction. Besides, with the second strategy (post-harvest treatments before consumption, such as boiling and vinegar dressing), both species showed Z enhancement. By combining both approaches in two-steps, the Z content can be enhanced up to 15-fold in spinach and 28-fold in rocket, increasing, as a result, the nutritional value of food., R.E. received a doctor specialization grant from the UPV/EHU and a JAE-Doctor-2011-046 grant from the CSIC. This research was supported by research BFU 2010-15021 from the Ministry of Education and Science of Spain and research project UPV/EHU-GVIT-624-13.