Potential Enhancement of Plant Iron Assimilation by Microbial-Induced Root Exudation of Phenolic Compounds

ABSTRACT Iron (Fe) deficiency in crop plants is a modern agricultural problem worldwide. Although multiple strategies have been evolved to improve Fe assimilation, some plant species, especially dicots and nongraminaceous monocots (strategy I plants), cannot avoid Fe deficiency in low Fe-availability soils. It is well documented that graminaceous plants (strategy II plants) employ the chelationbased Fe acquisition, and the strategy I plants use the reduction-based strategy to take up Fe. Intriguingly, under Fe deficiency the strategy I plants have recently been found to acquire Fe via exudation of phenolic compounds to mobilize Fe, which is much similar to the chelation-based mechanism of strategy II plants. Hitherto, increasing evidence has shown that soil microbes play a cooperative role in plant Fe acquisition. Several beneficial rhizobacteria have been found to increase plant Fe accumulation via activation of the reduction-based strategy. Moreover, microbial-induced root exudation of phenolic compounds can also promote plant Fe absorption by efficient mobilization of Fe, which increases Fe bioavailability in calcareous soils. Here, we briefly review the recent progress on the Fe assimilation strategies of strategy I and II plants, and further discuss the possible mechanisms underlying soil microbes enhance plant Fe acquisition

Unlocking Nutrient Potential: Harness The Power Of Micro-Nutrient Solubilizing Bacteria: A Review

Micro-nutrient solubilizing bacteria (MSB) play a pivotal role in the growthand developmentof plants by enhancing the availability of essential micronutrients in the soil. These bacteria possess unique capabilities to solubilizeotherwise unavailable forms of micro-nutrients, such as iron, zinc, copper,manganese, and others. As a result, they improve nutrient uptake, planthealth, and overall crop productivity. The use of MSB in agriculture canreduce reliance on chemical fertilizers, which can be costly and have negativeenvironmental impacts. By makingmicro-nutrients more available, MSB helpoptimize the use of existing soil nutrients.MSB canalso contribute to soilhealth and overall environmental sustainability. Additionally, MSB isadaptable to various soil types and climates, making them suitable for diverseagricultural settings. Their compatibility with sustainable practices alignswith efforts to promote environmentally friendly agricultural systems.Improving nutrient availability promotes balanced ecosystems and reducesthe risk of nutrient runoff, which can harm water bodies. Some MSB hasbeen reported to induce systemic resistance in plants against certainpathogens. They trigger the plant's defense mechanisms, making it moreresistant to diseases. This review aims to provide an in-depth understandingof the mechanisms through which MSBexert their beneficial effects on plantsand the potential implications for sustainable agriculture.It covers variousaspects of MSB, including their identification, functions, interactions withplants, environmental factors influencing their activity, and their applicationsin modern agricultur

Several Yeast Species Induce Iron Deficiency Responses in Cucumber Plants (Cucumis sativus L.)

Iron (Fe) deficiency is a first-order agronomic problem that causes a significant decrease in crop yield and quality. Paradoxically, Fe is very abundant in most soils, mainly in its oxidized form, but is poorly soluble and with low availability for plants. In order to alleviate this situation, plants develop different morphological and physiological Fe-deficiency responses, mainly in their roots, to facilitate Fe mobilization and acquisition. Even so, Fe fertilizers, mainly Fe chelates, are widely used in modern agriculture, causing environmental problems and increasing the costs of production, due to the high prices of these products. One of the most sustainable and promising alternatives to the use of agrochemicals is the better management of the rhizosphere and the beneficial microbial communities presented there. The main objective of this research has been to evaluate the ability of several yeast species, such as Debaryomyces hansenii, Saccharomyces cerevisiae and Hansenula polymorpha, to induce Fe-deficiency responses in cucumber plants. To date, there are no studies on the roles played by yeasts on the Fe nutrition of plants. Experiments were carried out with cucumber plants grown in a hydroponic growth system. The effects of the three yeast species on some of the most important Fe-deficiency responses developed by dicot (Strategy I) plants, such as enhanced ferric reductase activity and Fe2+ transport, acidification of the rhizosphere, and proliferation of subapical root hairs, were evaluated. The results obtained show the inductive character of the three yeast species, mainly of Debaryomyces hansenii and Hansenula polymorpha, on the Fe-deficiency responses evaluated in this study. This opens a promising line of study on the use of these microorganisms as Fe biofertilizers in a more sustainable and environmentally friendly agriculture

Induced Systemic Resistance (ISR) and Fe Deficiency Responses in Dicot Plants

Plants develop responses to abiotic stresses, like Fe deficiency. Similarly, plants also develop responses to cope with biotic stresses provoked by biological agents, like pathogens and insects. Some of these responses are limited to the infested damaged organ, but other responses systemically spread far from the infested organ and affect the whole plant. These latter responses include the Systemic Acquired Resistance (SAR) and the Induced Systemic Resistance (ISR). SAR is induced by pathogens and insects while ISR is mediated by beneficial microbes living in the rhizosphere, like bacteria and fungi. These root-associated mutualistic microbes, besides impacting on plant nutrition and growth, can further boost plant defenses, rendering the entire plant more resistant to pathogens and pests. In the last years, it has been found that ISReliciting microbes can induce both physiological and morphological responses to Fe deficiency in dicot plants. These results suggest that the regulation of both ISR and Fe deficiency responses overlap, at least partially. Indeed, several hormones and signaling molecules, like ethylene (ET), auxin, and nitric oxide (NO), and the transcription factor MYB72, emerged as key regulators of both processes. This convergence between ISR and Fe deficiency responses opens the way to the use of ISR-eliciting microbes as Fe biofertilizers as well as biopesticides. This review summarizes the progress in the understanding of the molecular overlap in the regulation of ISR and Fe deficiency responses in dicot plants. Root-associated mutualistic microbes, rhizobacteria and rhizofungi species, known for their ability to induce morphological and/or physiological responses to Fe deficiency in dicot plant species are also reviewed herei

Chapter 9 - Future Perspective in Organic Farming Fertilization: Management and Product

The concept of “organic farming” is based on a holistic view of farming systems, in which components are completely integrated into the system, such as the soil and its microbiota, climatic conditions, plants, and/or animals. The maintenance and the increase of fertility rely on the use of management practices and the application of different products from natural origins, which have the aim of providing high availability of nutrients for plant crops in agricultural soils. In this chapter, we will summarize the available information related to several inputs and practices allowed in the organic farming concept and vision, focusing on the benefits of their single or combined application. Moreover, we will introduce the latest novel applications of some of them, such as anaerobic digestion of manure, and present new alternatives, such as the use of biochar or the design of new biofertilization schemes

Entwicklung eines Beimpfungsverfahrens für Pflanzenkohle mit Bacillus subtilis

Die natürliche Heterogenität des Bodens ist das entscheidende Hindernis bei der Applikation pflanzen-nützlicher Organismen, weshalb eine Trägersubstanz benötigt wird, die den Zielorganismen ein geeignetes Mikrohabitat bieten kann. Heute wird dazu meist Torf verwendet, der aber unter starker Kritik steht. In den vergangenen Jahren wurden vermehrt Arbeiten publiziert, die Pflanzenkohle als Träger-mittel untersuchten. Demnach funktioniert die Beimpfung der Kohle, die Produkte können aber oft-mals nicht mit Torf als Trägersubstanz mithalten. Entsprechend sind weitere Optimierungen des Beimpfungsverfahrens notwendig. In dieser Arbeit wurde ein Beimpfungsverfahren für Pflanzenkohle mit Bacillus subtilis entwickelt, und eine Optimierung durch eine Nährstoffanreicherung und Oxidation der Pflanzenkohle überprüft. Zu-dem wurde in einem fünfwöchigen Haltbarkeitsversuch die Lagerfähigkeit von unbehandelter und oxidierter Pflanzenkohle, beimpft mit B. subtilis, ermittelt. In einem letzten Schritt wurde getestet, ob beimpfte Pflanzenkohle (unbehandelt und oxidiert) Pythium ultimum bei Kresse zu unterdrücken vermag und dadurch das Kresse-Wachstum fördern kann. Die Nährstoffanreicherung der Pflanzenkohle führte zu tieferen Zellzahlen im Inokulum, vermutlich weil sie eine Blockierung der Poren oder eine zu salzige Umgebung für B. subtilis verursachte. Im Ver-such zum Einfluss der Oxidation der Pflanzenkohle auf die Beimpfung konnte diese die resultierenden Zellzahlen signifikant erhöhen. In den folgenden Versuchen konnte die Wirkung aber nicht mehr nach-gewiesen werden und die Oxidation hatte weder einen Einfluss auf die Haltbarkeit der Formulierung noch auf die Wirkung gegen P. ultimum bei Kresse. Diese widersprüchlichen Ergebnisse sind vermutlich auf die Filtration während des Oxidierungsverfahrens zurückzuführen, denn erst in den Nachfolgeversuchen wurde auch die Kontrollkohle eingelegt und filtriert. Durch die Filtrierung wurden die kleinsten Kohlepartikel ausgewaschen, was zu einer höheren durchschnittlichen Porengrössenverteilung geführt haben könnte. Es ist anzunehmen, dass dadurch die Populationsgrösse auf der beimpften Kohle erhöht wurde und die Porengrössenverteilung daher einen entscheidenden Faktor bei der Beimpfung von Pflanzenkohle darstellt. Die koloniebildenden Einheiten auf der eingelagerten Pflanzenkohle waren nach fünf Wochen signifikant tiefer als zu Beginn und entsprachen nicht den Qualitätsstandards. Eine vorgängige Abfiltrierung könnte die Ergebnisse verbessern. Das Inokulum zeigte eine positive Wirkung auf das Kresse-Wachstum und konnte den Befall durch P. ultimum vermindern. Diese Wirkung ist allerdings stärker auf die Pflanzenkohleapplikation als auf B. subtilis zurückzuführen. Möglicherweise hat die Pflanzenkohle die Nährstoffversorgung oder die physikalischen Eigenschaften des Substrates verbessert und die Vitalität der Kresse dadurch gefördert. Zudem hat die Behandlung mit beimpfter Pflanzenkohle das Wachstum der Kresse im Vergleich zur Negativkontrolle nur leicht erhöht, was die Wichtigkeit einer weiteren Optimierung des Verfahrens hervorhebt. Die in dieser Arbeit auf der Pflanzen-kohle erreichten Populationsgrössen entsprachen den Qualitätsstandards, weshalb das angewendete Verfahren erfolgsversprechend für weitere Verbesserungen ist.The natural heterogeneity of soils is the key obstacle in the application of plant beneficial microorganisms, hence a carrier is needed that can provide a suitable microhabitat for the target organisms. Today, peat is generally the preferred material for this purpose, however it has been the subject of considerable criticism. In recent years, an increasing number of papers have been published that examined biochar as a new carrier substance. The inoculation of biochar has been successful, although the resulting products have not been able to compete with peat as a carrier substance in many cases. Accordingly, further optimisation of the inoculation process is needed. In this thesis, an inoculation process for biochar with Bacillus subtilis was developed and further an optimisation through the nutrient enrichment and oxidation of biochar was tested. In addition, the shelf life of untreated and oxidised charcoal inoculated with B. subtilis was determined in a five-week shelf life trial. In a final experiment, it was assessed whether inoculated charcoal (untreated and oxidised) is able to suppress Pythium ultimum in cress and thus promote cress growth. The nutrient enrichment of the biochar led to lower cell counts in the inoculum, presumably because it caused a blockage of pores or a too salty environment for B. subtilis. In the experiment assessing the influence of the biochar oxidation on the inoculation, oxidised biochar was able to host significantly higher cell counts. In the following tests, however, the effect could not be verified, and the oxidation had no influence on the shelf or the suppressive effect against P. ultimum in cress. These contradictory results are probably caused due to the filtration during the oxidation process, as only in the subsequent trials the control biochar was soaked and filtered as well. The filtration resulted in the leaching of the smallest biochar particles, which may have led to a higher average pore size distribution. It can be assumed that this process led to an increased population size on the inoculated biochar and hence the pore size distribution is a decisive factor in the inoculation process of biochar. The colony forming units on the inoculated biochar were significantly lower after five weeks of storage than at the beginning and did not meet the quality standards. Prior filtration may improve the results. The inoculum showed a positive effect on cress growth and was able to reduce infestation by P. ultimum. However, this effect was more strongly attributed to the biochar application than to B. subtilis. It might be that the biochar application has improved the nutrient supply or physical properties of the substrate and thus promoted the vitality of the cress. The treatment with inoculated biochar however increased the growth of cress only slightly compared to the negative control, which highlights the importance of further optimisation of the inoculation process. The population sizes achieved on the biochar in this thesis met the quality standards, thus the method used is promising for further improvements

Unravelling Micromonospora interactions with its host plant and the associated microbioma

[ES] La Unión Europea depende en gran medida de las importaciones de soja (> 70%) como fuente de proteínas, ya que la producción local apenas cubre el 5% de la demanda interna. Por ello, es necesario explorar fuentes alternativas para reducir esta dependencia. Entre las leguminosas, Lupinus angustifolius es una opción dado su alto valor proteico y su uso para la alimentación animal y humana. Esta leguminosa es una planta autóctona del continente europeo, que está bien adaptada a las condiciones climáticas de otras regiones como puede ser Australia o América. También crece de forma silvestre en suelos pobres gracias a su capacidad para fijar nitrógeno en simbiosis con bacterias. La adaptación de dicha planta puede deberse en parte a los microorganismos asociados a sus raíces, que le proporcionan estabilidad y resistencia, además de moléculas promotoras del crecimiento vegetal y nutrientes. Las comunidades microbianas asociadas a las plantas se ven influenciadas por diversos factores como son el genotipo/especie del huésped, el tipo de suelo, compartimento de la planta y estación climática, entre otros. Separar estos factores para saber cuáles son los que más influyen en la asociación de microorganismos a las plantas es una tarea muy complicada puesto que ninguno se da de forma independiente. En el primer capítulo de esta tesis doctoral, se abordó esta temática estudiando las variaciones estacionales y geográficas de la microbiota del suelo, y caracterizando el microbioma asociado a la planta Lupinus angustifolius en diferentes condiciones de cultivo mediante técnicas independientes de cultivo. En el segundo capítulo, el objetivo fue el aislamiento e identificación molecular de la comunidad bacteriana presente en los distintos tejidos de la planta y la generación de una colección de cepas asociada al microbioma de L. angustifolius. Con los resultados obtenidos en los dos primeros capítulos, se describió por primera vez el microbioma core de la planta L. angustifolius. En el tercer y último capítulo de esta tesis doctoral se trató de descifrar las interacciones de Micromonospora con su planta huésped y el microbioma asociado, empleando para tal fin la información obtenida en los capítulos anteriores. Se desarrollaron siete comunidades sintéticas que se inocularon en experimentos in planta, en condiciones de invernadero en un suelo con su comunidad natural, y en un sistema gnotobiótico con un sustrato estéril. Posteriormente se evaluó mediante técnicas independientes de cultivo cómo se ensamblaban los microorganismos a la raíz y cuál era el efecto de las distintas SynComs en la planta huésped y el microbioma circundante. [EN] The European Union highly depends on soy imports (> 70%) as a protein source since local production barely covers 5% of its internal demand. Thus, it is necessary to explore alternative sources to reduce this dependency. Among legumes, Lupinus angustifolius is an important alternative given its high protein value and use for animal and human nutrition. This legume is a native plant of Europe, well adapted to the climatic conditions of many countries. It also thrives in poor soils due to its capacity to fix nitrogen. Plant adaptation may be partly due to the microorganisms associated with its roots, providing stability and resilience, in addition to plant growth promoting molecules and nutrients. Plant-associated microbial communities are influenced by several factors such as host genotype/species, soil type, plant compartment and climatic season, among others. Separating these factors to understand which are the most influential in the association of microorganisms to plants is a very complex task as they do not occur independently. In the first chapter of this doctoral thesis, this topic was addressed by studying seasonal and geographical variations in the soil microbiota, and characterizing the microbiome associated with the plant Lupinus angustifolius under different cultivation conditions using an independent culture methodology. The results of the soil samples analysed suggest that the difference in the microbial community composition observed between the two sampling locations, Cabrerizos and Salamanca, was partly due to differences in soil conditions. None of the communities analysed (bacterial and fungal) showed differences in alpha diversity (Shannon index) between the climatic seasons in which the samples were collected. Beta diversity (Bray-Curtis-based principal coordinate analysis) for both microbial communities separated the samples into two groups according to soil type. In the case of bacteria, it was observed that, in addition, subgroups were formed according to the climatic seasons for the Salamanca soil. Interestingly, this also occurred with the fungal communities, where the samples were separated by season in both soil types. These results suggest that the main difference in soil microbial communities is due to edaphic properties, although environmental factors such as temperature, humidity or rainfall also influence the diversity of soil microbial communities. In addition, the microbiome associated with the legume Lupinus angustifolius cultivated under natural and greenhouse conditions was also characterized. For this purpose, wild and greenhouse-grown plants were collected from the same locations and analysed by 16S rRNA gene and ITS-2 gene profiling. Bacterial communities were characterized in the different plant compartments (rhizosphere, roots, nodules and leaves) while ITS profiles were restricted to the soil and rhizosphere. As previously reported for other plants, the highest richness was found in the rhizosphere, followed by the roots, leaves, and nodules. Within the rhizosphere, the bacterial richness in the in Salamanca plants was lower, especially for the field samples, probably affected by a pH below 7 and high amounts of P and K. In general, the compartments from the plants grown under greenhouse conditions showed a slightly higher bacterial diversity when compared to the wild plants. Within the fungal communities, the Shannon index was significantly higher in soil than rhizosphere samples (P1% and designed several isolation protocols. A total of 722 bacterial strains were isolated. As expected, the highest number of isolates was obtained in the rhizosphere compartment and a similar pattern was observed with a decreasing diversity gradient starting from the rhizosphere followed by the roots, leaves and nodules. In total, 87 different genera were identified, of which 19 had more than 10 isolates. The most abundant strains were identified in the genera Pseudomonas, Streptomyces, Agrobacterium, Bacillus and Pseudoclavibacter. In this work, 51.9% of the searched genera were isolated, and 74.7% of the isolated genera were identified by metagenomics, but 19.6% could not be detected in any plant compartment by metagenomics. Plant pathogenicity assays showed that 29% of the L. angustifolius isolates were potentially pathogenic for Arabidopsis thaliana Col-0. In turn, 394 strains (55%) were found to be non-pathogenic and 116 (16%) promoted the growth of A. thaliana. Analysis of metagenomics and culturomics results identified a core microbiome of the host plant L. angustifolius that included Acidovorax, Bradyrhizobium, Caulobacter, Chitinophaga, Flavobacterium, Kribella, Massilia, Pseudomonas, Pseudoxanthomonas, Rhizobium, Sphingomonas, Streptomyces and Variovorax. The composition and diversity of the identified host plant-associated bacteriome varied slightly between sampling locations and growing conditions. The genera identified as the core microbiome were present in more than 80% of the samples analysed. In chapter three of this work, the aim was to decipher the interactions of Micromonospora with its host plant and the associated microbiome, using the information obtained in the previous two chapters. Seven different synthetic communities (SynComs) were designed using bacterial strains isolated from the rhizosphere and roots of L. angustifolius to study their effect on the root and rhizosphere of the plant. In addition, we wanted to learn if the selected strains had any effect on the host plant and the natural bacterial communities present in the cultivation soils. After obtaining the genomes of the bacterial strains included in the different SynComs, a comparative genomic analysis was carried out, confirming that all the selected strains had genes with functions related to plant association and growth promotion. Plants were grown for 8 weeks in unsterilised soil under greenhouse conditions, and several plant parameters were measured and compared against the control plants (uninoculated). The plants inoculated with SynCom_7 showed the best growth and development. Furthermore, 16S rRNA gene profiling showed that the soil samples were the most diverse, followed by rhizosphere and roots (alpha diversity) (Figs. 54 and 55). Beta diversity grouped the samples into three clusters according to compartments: soil, rhizosphere and roots. In addition, a clustering pattern was observed for the SynComs inoculated in the root samples. All consortia that contained the nitrogen fixer, Bradyrhizobium sp. in the synthetic community formed one cluster, while the rest of the SynComs were recovered in a second cluster. The analysis of the bacterial composition of the bulk soil samples confirmed that the synthetic communities did not affect the composition of the soil where the plant was growing. However, when we studied the bacterial composition in the rhizosphere, a slight variation was observed, and the bacterial community of root samples was greatly influenced by the inoculated SynComs. The second part of this chapter consisted in the evaluation of the different SynComs on L. angustifolius plants grown in sterile soil under a gnotobiotic system. As in the first experiment, several growth parameters were registered, observing that plants inoculated with SynCom_7 showed the highest growths, again. Pseudomonas sp. Strain CRA141 showed the closest association with the roots. This result is not unexpected as it is well known that many Pseudomonas strains associate to plant roots. In addition, it was found that Micromonospora sp. Lupac 08 was detected in the rhizosphere and roots, and while this actinobacterium is not part of the core microbiome, it could be considered a "satellite" microorganism with important beneficial functions for the plant. Plant gene expression was related to the effect of the SynComs inoculated. When inoculated consortia included the Bradyrhizobium strain, very little differences were found when compared to the control plants, however, when only the Micromonospora strain and/or the other members of the SynComs were added, the differential gene expression increased threefold (Fig. 62). Gene ontology enrichment analyses revealed that those functions that were enriched by inoculating the different SynComs were clearly related to plant-microbe interaction functions. The same was observed for the enriched metabolic pathways when KEGG analysis was performed

Augmenting Iron Accumulation in Cassava by the Beneficial Soil Bacterium Bacillus subtilis (GBO3)

Cassava (Manihot esculenta), a major staple food in the developing world, provides a basic carbohydrate diet for over half a billion people living in the tropics. Despite the iron abundance in most soils, cassava provides insufficient iron for humans as the edible roots contain 3-12 times less iron than other traditional food crops such as wheat, maize, and rice. With the recent identification that the beneficial soil bacterium Bacillus subtilis (strain GB03) activates iron acquisition machinery to increase metal ion assimilation in Arabidopsis, the question arises as to whether this plant-growth promoting rhizobacterium (PGPR) also augments iron assimilation to increase endogenous iron levels in cassava. Biochemical analyses reveal that shoot-propagated cassava with GB03-inoculation exhibit elevated iron accumulation after 140 days of plant growth as determined by X-ray microanalysis and total foliar iron analysis. Growth promotion and increased photosynthetic efficiency were also observed for greenhouse-grown plants with GB03-exposure. These results demonstrate the potential of microbes to increase iron accumulation in an important agricultural crop and is consistent with idea that microbial signaling can regulate plant photosynthesis

Role of ethylene and other signals in the regulation of responses to Iron, Phosphorus or Sulfur deficiencies

Para su correcto crecimiento y desarrollo, las plantas necesitan diversos nutrientes minerales, entre los que se encuentran los elementos objeto de estudio de esta Tesis: hierro (Fe), fósforo (P) y azufre (S). Estos elementos son esenciales para las plantas y, aunque suelen ser abundantes en la mayoría de los suelos, su disponibilidad para las plantas puede ser baja, sobre todo en determinados suelos, como los calcáreos. Para facilitar su adquisición, las plantas han desarrollado diferentes mecanismos. En el caso del hierro, y dependiendo del tipo de mecanismos, las plantas se dividen en plantas de Estrategia I (todas las plantas excepto las gramíneas) y plantas de Estrategia II (gramíneas). En condiciones de deficiencia de alguno de estos nutrientes, las plantas dicotiledóneas (con Estrategia I y objeto de esta Tesis) ponen en marcha una serie de respuestas, principalmente en sus raíces, para mejorar la movilización y transporte del nutriente en cuestión. Entre estas respuestas, encontramos cambios morfológicos de la raíz, como el desarrollo de pelos radicales subapicales y raíces proteoides (en respuesta a las deficiencias de hierro o fósforo), o un incremento del desarrollo de raíces laterales (en respuesta a las deficiencias de hierro, fósforo o azufre). También ocurren respuestas fisiológicas, como el incremento del número de transportadores, el incremento de la síntesis y liberación de compuestos que solubilizan los nutrientes en el suelo, y el incremento de algunas actividades enzimáticas, como la actividad de la reductasa férrica (en respuesta a la deficiencia de hierro) o de la fosfatasa ácida (en respuesta a la deficiencia de fósforo). Una vez el nutriente se ha adquirido en suficiente cantidad, estas respuestas deben ser inhibidas para minimizar el coste energético y evitar la toxicidad por exceso, lo que implica que estas respuestas deben estar sujetas a un control muy estricto. En la regulación de estas respuestas, el etileno juega un papel fundamental. Por ello, uno de los objetivos principales de esta Tesis ha sido profundizar en el papel de la proteína EIN2, clave en la ruta de señalización del etileno, en la regulación de las respuestas a la deficiencia de hierro. Este objetivo se abordó en el primer trabajo de esta Tesis “Comparative study of several Fe deficiency responses in the Arabidopsis thaliana ethylene insensitive mutants ein2-1 and ein2-5”, publicado en la revista Plants. Aunque las respuestas a cada deficiencia se inducen de manera específica, es frecuente que ocurra un cierto cruce de respuestas, de manera que, a veces, la deficiencia de un nutriente puede inducir respuestas relacionadas con las deficiencias de otros nutrientes. En la regulación de las respuestas a las deficiencias de hierro, fósforo o azufre, se ha implicado al etileno. Esa común implicación podría explicar parcialmente la inducción cruzada de respuestas a las distintas deficiencias. Para conferir especificidad, el etileno podría actuar junto con otras señales y/o a través de diferentes rutas de señalización para la regulación de las distintas respuestas a las diferentes deficiencias. En este sentido, uno de los principales objetivos de esta Tesis ha sido tratar de esclarecer la participación de algunos de los componentes clave de la ruta de señalización del etileno en la regulación de las respuestas a las deficiencias de hierro, fósforo o azufre. Para ello, se realizaron ensayos con distintos mutantes de Arabidopsis thaliana, afectados en alguno de los componentes de la ruta de señalización del etileno, y se sometieron a deficiencia de hierro, fósforo o azufre. A continuación, se estudiaron respuestas específicas a cada una de las tres deficiencias, así como respuestas a las otras deficiencias, en cada uno de estos mutantes. Estos estudios nos permitieron conocer qué elementos de la ruta de señalización del etileno son claves para cada una de las respuestas y a qué nivel se produce la interacción entre ellas Los resultados obtenidos se han publicado en Frontiers in Plant Science “Influence of ethylene signaling in the crosstalk between Fe, S, and P deficiency responses in Arabidopsis thaliana”. Por último, el tercer gran objetivo de esta Tesis ha sido tratar de dilucidar la posible relación existente entre distintas señales reguladoras de las respuestas a la deficiencia de hierro ya conocidas, como es el caso del etileno y de LODIS (‘LOng Distance Iron Signal’; señal represiva de las respuestas relacionada con el contenido interno de hierro), y unos pequeños péptidos (IMA1, 2 y 3), recientemente identificados en Arabidopsis y que actúan como activadores de las respuestas a la deficiencia de hierro. Nuestro objetivo ha sido tratar de encajar los péptidos IMA en el modelo de regulación existente. Los resultados obtenidos relacionados con este objetivo se han publicado también en Frontiers in Plant Science “A shoot derived long distance iron signal may act upstream of the IMA peptides in the regulation of Fe deficiency responses in Arabidopsis thaliana roots”.For their correct growth and development, plants need various mineral nutrients, like the elements studied in this Thesis: iron (Fe), phosphorus (P) and sulfur (S). These elements are essential for plants and, although they are usually abundant in most soils, their availability for plants can be low, especially in certain soils, such as the calcareous ones. To facilitate their acquisition, plants have developed different mechanisms. In the case of iron, based on the acquisition mechanisms, plants are divided into Strategy I plants (all plants except grasses) and Strategy II plants (grasses). Under nutrient deficiency conditions, dicotyledonous plants (with Strategy I and object of this Thesis) induce several responses, mainly in their roots, to improve the mobilization and transport of the deficient nutrient. Among these responses, there are morphological changes of the root, such as the development of subapical root hairs and proteoid roots (in response to iron or phosphorous deficiencies), or an increase in lateral root development (in response to iron, phosphorus or sulfur deficiencies). Physiological responses also occur, such as an increase of nutrient transporters, an increase in the synthesis and release of several compounds that solubilize nutrients in the soil, and an increase in some enzymatic activities, such as ferric reductase activity (in response to iron deficiency) or acid phosphatase (in response to phosphorus deficiency). Once the nutrient has been acquired in sufficient quantity, these responses must be inhibited to minimize energy cost and to avoid toxicity by excess. This implies that the responses must be subjected to very strict control. In the regulation of these responses, ethylene plays a key role. For this reason, one of the main objectives of this Thesis has been to look further into the role of the EIN2 protein, a key component of the ethylene signaling pathway, in the regulation of the iron deficiency responses. This objective has been addressed in the first work of this Thesis "Comparative study of several Fe deficiency responses in the Arabidopsis thaliana ethylene insensitive mutants ein2-1 and ein2-5", published in the journal Plants. Although responses to each deficiency are induced in a specific manner, some crosstalk among the responses to different nutrient deficiencies often occur. This implies that, sometimes, the typical responses to a nutrient deficiency can be induced by other nutrient deficiencies. Ethylene has been involved in the regulation of the iron, phosphorous or sulfur deficiency responses. This common implication could partially explain the crosstalk of the responses to these different deficiencies. To confer specificity, ethylene could act in conjunction with other signals, and/or through different signaling pathways, to regulate different responses to different deficiencies. In this sense, one of the main objectives of this Thesis has been to clarify the participation of some of the key components of the ethylene signaling pathway in the regulation of the responses to iron, phosphorus or sulfur deficiencies. For this, several experiments were carried out with Arabidopsis thaliana mutants, affected in some components of the ethylene signaling pathway, and subjected to iron, phosphorus or sulfur deficiencies. Next, specific responses to each deficiency, as well as responses to the other deficiencies, in each of these mutants were studied. These studies allowed us to find out which elements of the ethylene signaling pathway play key roles in each response and the interactions between them. The results obtained have been published in Frontiers in Plant Science "Influence of ethylene signaling in the crosstalk between Fe, S, and P deficiency responses in Arabidopsis thaliana”. Finally, the third major objective of this Thesis has been to elucidate the possible connection between different regulatory signals of the iron deficiency responses already known, such as ethylene and LODIS ('LOng Distance Iron Signal'; repressive signal of responses related to internal iron content), and some small peptides (IMA1, 2 and 3), recently identified in Arabidopsis that act as activators of the iron deficiency responses. Our objective has been to fit the IMA peptides into the existing regulatory model. The results obtained related to this objective have also been published in Frontiers in Plant Science “A shoot derived long distance iron signal may act upstream of the IMA peptides in the regulation of Fe deficiency responses in Arabidopsis thaliana roots”