Acumulación de ureidos en respuesta al déficit hídrico en Pahseolus vulgaris y caracterización de la PRAT y la XDH, dos enzimas clave en la síntesis de ureidos

Los ureidos alantoína y alantoato son las formas mayoritarias en las que se almacena y transporta el nitrógeno fijado en los nódulos de las leguminosas tropicales o de tipo ureídico como la soja (Glycine max) y la judía (Phaseolus vulgaris). En las leguminosas ureídicas, el déficit hídrico produce la inhibición de la fijación de nitrógeno, a la vez que aumenta el contenido de ureidos en las hojas. La síntesis de ureidos se lleva a cabo por el catabolismo oxidativo de purinas que pueden haber sido sintetizadas de novo o proceder del reciclaje de los nucleótidos de purina. En este trabajo, se ha llevado a cabo un análisis del metabolismo de los ureidos en variedades de P. vulgaris con distinto grado de tolerancia a la sequía. La determinación del contenido de ureidos en los distintos genotipos de judía sometidos a estrés hídrico mostró que la acumulación de ureidos se correlacionaba con el grado de tolerancia a la sequía de los mismos, siendo mayor en los más sensibles. Además, la acumulación de ureidos se debía a la activación de su síntesis en tejidos diferentes a los nódulos. Para analizar la procedencia de los ureidos acumulados durante el estrés hídrico, se han estudiado las enzimas xantina deshidrogenasa (XDH) y glutamina fosforribosilpirofosfato amidotransferasa (PRAT), cruciales en la regulación de la oxidación y en la síntesis de novo de purinas, respectivamente. Se ha aislado la secuencia codificante y la región promotora del gen XDH de P. vulgaris, en la que se han encontrado diversos motivos de regulación por estrés. Los análisis de expresión y de actividad mostraron un aumento del nivel de PvXDH en respuesta a la sequía y a tratamientos con diferentes fitohormonas relacionadas con estreses abióticos o bióticos. Además de la actividad deshidrogenasa, la enzima PvXDH es capaz de catalizar la oxidación de la xantina y el NADH por oxígeno molecular. Sin embargo, solamente la actividad deshidrogenasa aumentó por el estrés. Curiosamente, el óxido nítrico (ON) produjo una inhibición de la actividad XDH en las hojas pero no en los nódulos de judía y la inhibición en hojas se impidió por ácido úrico añadido. Cuando se bloqueó la expresión del gen PvXDH por...The ureides allantoin and allantoate are the mayor forms in which the nitrogen fixed in the nodules is stored and exported in tropical or ureidic legumes such as the soybean (Glycine max) and common bean (Phaseolus vulgaris). In ureidic legumes, nitrogen fixation is inhibited under water deficit, but the ureide content of leaves increases under these conditions. Ureide synthesis takes place by the oxidative degradation of purine bases, whose origin may be the de novo synthesis or the remobilization of preexisting purine nucleotides. In this work, an analysis of the ureide metabolism has been performed in several varieties of P. vulgaris with different degree of drought tolerance. The determination of the ureide concentration in different common bean genotypes subjected to water stress showed that the accumulation of ureides correlates with the degree of drought sensitivity of the varieties, being higher in the sensitive varieties than in the tolerant ones. Moreover, ureides accumulation was due to the activation of their synthesis in tissues other than nodules. To determine the origin of ureides accumulated during water deficit, the enzymes xanthine dehydrogenase (XDH) and glutamine phosphoribosylpyrophosphate amidotransferase (PRAT), which are pivotal in the regulation of the oxidation and de novo synthesis of purines, respectively, have been studied. The coding sequence and the promoter region of the XDH gene from P. vulgaris have been isolated. Analysis of the sequences revealed the presence of several stress-related regulatory motifs in the proximal promoter sequence and expression and activity analyses showed an enhancement in the PvXDH level in response to drought and in response to treatments with phytohormones related to abiotic and biotic stresses. Besides the dehydrogenase activity, the PvXDH protein is also able to catalyze the oxidation of xanthine and NADH by molecular oxygen; however, only the dehydrogenase activity increased in response to stress. Interestingly, nitric oxide (NO) inhibited the XDH activity in leaves, but not in nodules of common bean, and the inhibition in leaves was blocked by exogenous uric acid. When the expression of PvXDH gene was..

New insights on arabidopsis thaliana root adaption to ammonium nutrition by the use of a quantitative proteomic approach

Nitrogen is an essential element for plant nutrition. Nitrate and ammonium are the two major inorganic nitrogen forms available for plant growth. Plant preference for one or the other form depends on the interplay between plant genetic background and environmental variables. Ammonium-based fertilization has been shown less environmentally harmful compared to nitrate fertilization, because of reducing, among others, nitrate leaching and nitrous oxide emissions. However, ammonium nutrition may become a stressful situation for a wide range of plant species when the ion is present at high concentrations. Although studied for long time, there is still an important lack of knowledge to explain plant tolerance or sensitivity towards ammonium nutrition. In this context, we performed a comparative proteomic study in roots of Arabidopsis thaliana plants grown under exclusive ammonium or nitrate supply. We identified and quantified 68 proteins with differential abundance between both conditions. These proteins revealed new potential important players on root response to ammonium nutrition, such as H+ -consuming metabolic pathways to regulate pH homeostasis and specific secondary metabolic pathways like brassinosteroid and glucosinolate biosynthetic pathways

Knock-down of phosphoenolpyruvate carboxylase 3 negatively impacts growth, productivity, and responses to salt stress in sorghum (Sorghum bicolor L.)

Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabolism. Most studies in C-4 plants have focused on photosynthetic PEPC, but less is known about non-photosynthetic PEPC isozymes, especially with respect to their physiological functions. In this work, we analyzed the precise roles of the sorghum (Sorghum bicolor) PPC3 isozyme by the use of knock-down lines with the SbPPC3 gene silenced (Ppc3 lines). Ppc3 plants showed reduced stomatal conductance and plant size, a delay in flowering time, and reduced seed production. In addition, silenced plants accumulated stress indicators such as Asn, citrate, malate, and sucrose in roots and showed higher citrate synthase activity, even in control conditions. Salinity further affected stomatal conductance and yield and had a deeper impact on central metabolism in silenced plants compared to wild type, more notably in roots, with Ppc3 plants showing higher nitrate reductase and NADH-glutamate synthase activity in roots and the accumulation of molecules with a higher N/C ratio. Taken together, our results show that although SbPPC3 is predominantly a root protein, its absence causes deep changes in plant physiology and metabolism in roots and leaves, negatively affecting maximal stomatal opening, growth, productivity, and stress responses in sorghum plants. The consequences of SbPPC3 silencing suggest that this protein, and maybe orthologs in other plants, could be an important target to improve plant growth, productivity, and resistance to salt stress and other stresses where non-photosynthetic PEPCs may be implicated.This research was supported by the Junta de Andalucía (P12-FQM-489 and PAI group BIO298), the Basque Government (IT932-16), and the Ministerio de Economía, Industria y Competitividad (AGL2012-35708 and AGL2016-75413-P)

Molecular and biochemical analysis of XDH from Phaseolus vulgaris suggest that uric acid protects the enzyme against the inhibitory effects of nitric oxide in nodules

Corrigendum of “Molecular and biochemical analysis of XDH from Phaseolus vulgaris suggest that uric acid protects the enzyme against the inhibitory effects of nitric oxide in nodules” [Plant Physiol. Biochem. 143 (2019) 364–374]Xanthine dehydrogenase (XDH) is essential for the assimilation of symbiotically fixed nitrogen in ureidic legumes. Uric acid, produced in the reaction catalyzed by XDH, is the precursor of the ureides, allantoin and allantoate, which are the main N-transporting molecules in these plants. XDH and uric acid have been reported to be involved in the response to stress, both in plants and animals. However, the physiological role of XDH under stressful conditions in ureidic legumes remains largely unexplored. In vitro assays showed that Phaseolus vulgaris XDH (PvXDH) can behave as a dehydrogenase or as an oxidase. Therefore, it could potentially protect against oxidative radicals or, in contrast, it could increase their production. In silico analysis of the upstream genomic region of XDH coding gene from P. vulgaris revealed the presence of several stress-related cis-regulatory elements. PvXDH mRNA and enzymatic activity in plants treated with stress-related phytohormones or subjected to dehydration and stressful temperatures showed several fold induction. However, PvXDH activity was in vivo and in vitro inhibited by nitric oxide in leaves but not in nodules. In extracts from RNAi PvXDH silenced nodules, with lower levels of uric acid, XDH activity was inhibited by SNP which indicates that uric acid produced by XDH in the nodules of this ureidic legume could help to protect XDH against the inhibitory effects of nitric oxide.This work was supported by Grants: AGL2012-34230 (Ministerio de Economia y Competitividad, Spain), AGL2015-69554-P (Ministerio de Economia, Industria y Competitividad, Spain) and P07-RNM-03307 and BIO-115 (Consejeria de Economia, hmovacion, Ciencia, Junta de Andalucia, Spain). I. Coleto was supported by FPU Fellowship (Ministerio de Ciencia e Innovacion, Spain)

<i>Arabidopsis thaliana</i> transcription factors <i>MYB28</i> and <i>MYB29</i> shape ammonium stress responses by regulating Fe homeostasis

15 Pág.Although ammonium (NH4+ ) is a key intermediate of plant nitrogen metabolism, high concentrations of NH4+ in the soil provoke physiological disorders that lead to the development of stress symptoms. Ammonium nutrition was shown to induce the accumulation of glucosinolates (GSLs) in leaves of different Brassicaceae species. To further understand the link between ammonium nutrition and GSLs, we analysed the ammonium stress response of Arabidopsis mutants impaired in GSL metabolic pathway. We showed that the MYB28 and MYB29 double mutant (myb28myb29), which is almost deprived of aliphatic GSLs, is highly hypersensitive to ammonium nutrition. Moreover, we evidenced that the stress symptoms developed were not a consequence of the lack of aliphatic GSLs. Transcriptomic analysis highlighted the induction of an iron (Fe) deficiency response in myb28myb29 under ammonium nutrition. Consistently, ammonium-grown myb28myb29 plants showed altered Fe accumulation and homeostasis. Interestingly, we showed overall that growing Arabidopsis with increased Fe availability relieved ammonium stress symptoms and that this was associated with MYB28 and MYB29 expression. Taken together, our data indicated that the control of Fe homeostasis was crucial for the Arabidopsis response to ammonium nutrition and evidenced that MYB28 and MYB29 play a role in this control.The research leading to these results has received funding from the Basque Government, IT-932-16 (IC, IB, DM) and the Spanish Government, BIO2014-56271-R and BIO2017-84035-R, both co-funded by FEDER (IC, IB, DM). IB holds a fellowship from the Basque Government. This work was further supported by the Danish National Research Foundation, DNRF grant 99 (MB) and the Danish Council for Independent Research, DFF-4181-00077 (CR, MB). This work was also supported by the National Institute for Agriculture and Food Research and Technology, RTA2015-00014-c02-01 (JM). We thank John Celenza for providing seeds for the cyp79b2b3 mutant, George Jander for the tgg1tgg2 mutant, Masami Hirai for the myb28myb29 mutant and Andrea Chini for the myc234 mutant. The authors thank the technical and human support provided by SGIker (UPV/EHU).Peer reviewe

Isotopic labelling reveals the efficient adaptation of wheat root TCA cycle flux modes to match carbon demand under ammonium nutrition

Proper carbon (C) supply is essential for nitrogen (N) assimilation especially when plants are grown under ammonium (NH4+) nutrition. However, how C and N metabolic fluxes adapt to achieve so remains uncertain. In this work, roots of wheat (Triticum aestivum L.) plants grown under exclusive NH4+ or nitrate (NO3-) supply were incubated with isotope-labelled substrates ((NH4+)-N-15, (NO3-)-N-15, or [C-13]Pyruvate) to follow the incorporation of N-15 or C-13 into amino acids and organic acids. Roots of plants adapted to ammonium nutrition presented higher capacity to incorporate both (NH4+)-N-15 and (NO3-)-N-15 into amino acids, thanks to the previous induction of the NH4+ assimilative machinery. The N-15 label was firstly incorporated into [N-15]Gln via glutamine synthetase; ultimately leading to [N-15]Asn accumulation as an optimal NH4+ storage. The provision of [C-13]Pyruvate led to [C-13]Citrate and [C-13] Malate accumulation and to rapid [C-13]2-OG consumption for amino acid synthesis and highlighted the importance of the anaplerotic routes associated to tricarboxylic acid (TCA) cycle. Taken together, our results indicate that root adaptation to ammonium nutrition allowed efficient assimilation of N thanks to the promotion of TCA cycle open flux modes in order to sustain C skeleton availability for effective NH4+ detoxification into amino acids.The research leading to these results has received funding from the Basque Government (IT-932-16), the Spanish Government (AGL2015-64582-C3-2-R MINECO/FEDER) and the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement number 334019. The authors thank the technical and human support provided by Phytotron Service (SGIker, UPV/EHU)

Conceptions of Crisis Management Capabilities – Municipal Officials’ Perspectives

In the Swedish crisis management system, the municipalities have a great responsibility. One part of this responsibility concerns preparing for crises by making risk and vulnerability analyses as well as plans for how to handle extraordinary events. Such preparedness planning involves municipal officials and consequently their conceptions of their organisations’ crisis management capabilities. This makes it vital to look into these conceptions more closely and establish whether specific characteristics can be identified. This thesis aims at gaining understanding of how officials involved in preparedness planning in general and vulnerability analysis in particular explicitly conceive of their organisations’ crisis management capabilities. The thesis poses six specific research questions, pertaining to three themes: vulnerability, dependencies and learning. The results show specific characteristics in how officials conceive of their organisations’ crisis management capabilities. These characteristics appear as similarities, variations, and even disagreements. It is argued that the characteristics as well as what explains them must be considered in the development of society’s crisis management systems