Polyamine metabolism and signaling in plant abiotic stress protection

Polyamines (PAs) are small polycationic compounds present in all living organisms. Compelling evidences indicate a role for PAs in plant protection against stress. During the recent years, genetic, molecular and ‘omic’ approaches have been undertaken to unravel the role of PAs in stress signaling. Overall, results point to intricate relationships between PAs, stress hormone pathways and ROS signaling. Such cross-regulations condition stress signaling through the modulation of abscisic acid (ABA) and ROS amplification-loops. In this chapter we compile our recent findings which elucidate molecular mechanisms and signalingpathways by which PAs contribute to stress protection in plants.

Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Background Plant hormones play a pivotal role in several physiological processes during a plant's life cycle, from germination to senescence, and the determination of endogenous concentrations of hormones is essential to elucidate the role of a particular hormone in any physiological process. Availability of a sensitive and rapid method to quantify multiple classes of hormones simultaneously will greatly facilitate the investigation of signaling networks in controlling specific developmental pathways and physiological responses. Due to the presence of hormones at very low concentrations in plant tissues (10-9 M to 10-6 M) and their different chemistries, the development of a high-throughput and comprehensive method for the determination of hormones is challenging. Results The present work reports a rapid, specific and sensitive method using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) to analyze quantitatively the major hormones found in plant tissues within six minutes, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all plant hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of plant material without time-consuming additional steps such as purification, sample drying or re-suspension. Conclusions This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study plant developmental processes or plant responses to biotic and abiotic stresses in complex tissues. An example is shown in which a hormone profiling is obtained from leaves of plants exposed to salt stress in the aromatic plant, Rosmarinus officinalis.

Modulation of plant HMG-CoA reductase by protein phosphatase 2A: Positive and negative control at a key node of metabolism

The enzyme HMG-CoA reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis, critical not only for normal plant development, but also for the adaptation to demanding environmental conditions. Consistent with this notion, plant HMGR is modulated by many diverse endogenous signals and external stimuli. Protein phosphatase 2A (PP2A) is involved in auxin, abscisic acid, ethylene and brassinosteroid signaling and now emerges as a positive and negative multilevel regulator of plant HMGR, both during normal growth and in response to a variety of stress conditions. The interaction with HMGR is mediated by B" regulatory subunits of PP2A, which are also calcium binding proteins. The new discoveries uncover the potential of PP2A to integrate developmental and calcium-mediated environmental signals in the control of plant HMGR.

Poliamines en estrès abiòtic i mecanismes moleculars

Les poliamines (PAs) putrescina (Put), espermidina (Spd) i espermina (Spm) són mol•lècules policatiòniques de baix pes molecular, presents en els microorganismes, animals i plantes. Les PAs han estat implicades en diversos processos cel•lulars importants, incloent la resposta de les plantes a l'estrès. No obstant això, el seu mode d'acció està es desconeix. En les plantes, es van acumulant evidències de que les PAs interactuen amb macromolècules i estructures cel•lulars, com ara proteïnes de membrana, i la seva possible participació en transducció de senyals s'ha convertit en una creixent i interessant àrea d'estudi. En aquesta tesi, la possible interacció entre les poliamines i les vies de senyalització de fosfolípids és investigada. Resultats previs, han posat de manifest que alteracions en els nivells endògens de poliamines (PAs), per sobreexpressió gènica o pèrdua de funció de gens de biosíntesi, redueixen o milloren, respectivament, la capacitat de les plantes d'Arabidopsis per tolerar agressions per estrès abiòtic, produint en alguns casos notables alteracions en el desenvolupament. En aquestes plantes amb nivells alterats de PAs s'han detectat canvis importants en l'expressió gènica i s'ha trobat una connexió entre el contingut de PAs ii la biosíntesi / senyalització d'àcid abscísic (ABA). La hipòtesi actual de treball és que aquestes alteracions en l'expressió gènica poden estar mediades, si més no en part, pel catabolisme de PAs, i l’acció directa o indirecta de les espècies reactives d'oxigen (ROS) que se’n deriven.

Teleost fish larvae adapt to dietary arachidonic acid supply through modulation of the expression of lipid metabolism and stress response genes

Dietary fatty acid supply can affect stress response in fish during early development. Although knowledge on the mechanisms involved in fatty acid regulation of stress tolerance is scarce, it has often been hypothesised that eicosanoid profiles can influence cortisol production. Genomic cortisol actions are mediated by cytosolic receptors which may respond to cellular fatty acid signalling. An experiment was designed to test the effects of feeding gilthead sea-bream larvae with four microdiets, containing graded arachidonic acid (ARA) levels (0·4, 0·8, 1·5 and 3·0 %), on the expression of genes involved in stress response (steroidogenic acute regulatory protein, glucocorticoid receptor and phosphoenolpyruvate carboxykinase), lipid and, particularly, eicosanoid metabolism (hormone-sensitive lipase, PPARα, phospholipase A2, cyclo-oxygenase-2 and 5-lipoxygenase), as determined by real-time quantitative PCR. Fish fatty acid phenotypes reflected dietary fatty acid profiles. Growth performance, survival after acute stress and similar whole-body basal cortisol levels suggested that sea-bream larvae could tolerate a wide range of dietary ARA levels. Transcription of all genes analysed was significantly reduced at dietary ARA levels above 0·4 %. Nonetheless, despite practical suppression of phospholipase A2 transcription, higher leukotriene B4 levels were detected in larvae fed 3·0 % ARA, whereas a similar trend was observed regarding PGE2 production. The present study demonstrates that adaptation to a wide range of dietary ARA levels in gilthead sea-bream larvae involves the modulation of the expression of genes related to eicosanoid synthesis, lipid metabolism and stress response. The roles of ARA, other polyunsaturates and eicosanoids as signals in this process are discussed.

Protein phosphatase inhibition assays for okadaic acid detection in shellfish: matrix effects, applicability and comparison with LC-MS/MS analysis

The applicability of the protein phosphatase inhibition assay (PPIA) to the determination of okadaic acid (OA) and its acyl derivatives in shellfish samples has been investigated, using a recombinant PP2A and a commercial one. Mediterranean mussel, wedge clam, Pacific oyster and flat oyster have been chosen as model species. Shellfish matrix loading limits for the PPIA have been established, according to the shellfish species and the enzyme source. A synergistic inhibitory effect has been observed in the presence of OA and shellfish matrix, which has been overcome by the application of a correction factor (0.48). Finally, Mediterranean mussel samples obtained from Rı´a de Arousa during a DSP closure associated to Dinophysis acuminata, determined as positive by the mouse bioassay, have been analysed with the PPIAs. The OA equivalent contents provided by the PPIAs correlate satisfactorily with those obtained by liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Lactic acid bacteria from fresh fruit and vegetables as biocontrol agents of phytopathogenic bacteria and fungi

This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 LAB strains was tested in vitro and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also analyzed for the inhibition of P. expansum infection in wounds of Golden Delicious apples. Four strains (TC97, AC318, TM319, and FF441) reduced the fungal rot diameter of the apples by 20%; only Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred mediators of inhibition but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against E. carotovora. While previous reports of antifungal activity by LAB are scarce, our results support the potential of LAB as biocontrol agents against postharvest rot. [Int Microbiol 2008; 11(4):231-236]

Conjugation of genetically-engineered protein phosphatases to magnetic particles for okadaic acid detection

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3μg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1μg/L).

Comparative study of algal communities in acid and alkaline waters from Tinto, Odiel and Piedras river basins (SW Spain)

The distribution patterns of benthic algal assemblages in the Tinto, Odiel and Piedras rivers were analyzed during the winter of 2005 in 18 sampling stations. The main objective was to assess and compare the algal communities and parameters affecting them both in the zones affected by acid mine drainage (AMD) and in naturally alkaline waters. A total of 108 benthic diatom taxa and 31 non-diatom taxa were identi ed. Results showed large differences between algal communities in the two environments: Pinnularia acoricola, P. subcapitata and Eunotia exigua were the most frequent diatom taxa in regions affected by acid mine drainage, along with algae like Klebsormidium and Euglena mutabilis were the most relevant non-diatom taxa. In alkaline waters the dominant diatom taxa were Planothidium frequentissimum, Gomphonema angustum, Fragilaria capucina, and some species of Navicula (N. viridula, N. veneta or N. radiosa), accompanied by Oscillatoria and Anabaena as well as by streptophytes of the group of zygnemataceae and desmidiaceae

Prediction of subcutaneous fatty acid composition from ham of CLA fed boars and gilts using a NIRS fiber optic probe

The aim of this work was the use of NIR technology by direct application of a fiber optic probe on back fat to analyze the fatty acid composition of CLA fed boars and gilts. 265 animals were fed 3 different diets and the fatty acid profile of back fat from Gluteus medius was analyzed using gas chromatography and FT-NIR. Spectra were acquired using a Bruker Optics Matrix-F duplex spectrometer equipped with a fiber optic probe (IN-268-2). Oleic and stearic fatty acids were predicted accurately; myristic, vaccenic and linoleic fatty acids were predicted with lower accuracy, while palmitic and α-linolenic fatty acids were poorly predicted. The relative percentage of fatty acids and NIR spectra showed differences in fatty acid composition of back fat from pigs fed CLA which increased the relative percentage of SFA and PUFA while MUFA decreased. Results suggest that a NIR fiber optic probe can be used to predict total saturated and unsaturated fatty acid composition, as well as the percentage of stearic and oleic. NIR showed potential as a rapid and easily implemented method to discriminate carcasses from animals fed different diets.

Del Aprendizaje Basado En Problemas (ABP) al Aprendizaje Basado En La Acción (ABA). Claves para su complementariedad e implementación

Estudios previos han sugerido que a pesar de las innumerables ventajas que ofrece la metodología activa de enseñanza-aprendizaje ABP (“Aprendizaje Basado en Problemas”), también presenta, como toda estrategia educativa, ciertas limitaciones. En este sentido, se ha sugerido que en muchas ocasiones las “situaciones” (“problemas”) que se utilizan están descontextualizadas, siendo artificiales y poco vinculadas con la acción real en contextos reales. Con el objetivo de superar estas limitaciones, se propone el “Aprendizaje Basado en la Acción” (ABA) como estrategia complementaria. El artículo, siguiendo el marco teórico de la psicología cultural de orientación vygotskiana, articula la propuesta e ilustra su aplicación

Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1.

The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.

Mitochondrial Fatty Acid Oxidation in Obesity

Significance: Current lifestyles with high-energy diets and little exercise are triggering an alarming growth in obesity. Excess of adiposity is leading to severe increases in associated pathologies, such as insulin resistance, type 2 diabetes, atherosclerosis, cancer, arthritis, asthma, and hypertension. This, together with the lack of efficient obesity drugs, is the driving force behind much research. Recent Advances: Traditional anti-obesity strategies focused on reducing food intake and increasing physical activity. However, recent results suggest that enhancing cellular energy expenditure may be an attractive alternative therapy. Critical Issues: This review evaluates recent discoveries regarding mitochondrial fatty acid oxidation (FAO) and its potential as a therapy for obesity. We focus on the still controversial beneficial effects of increased FAO in liver and muscle, recent studies on how to potentiate adipose tissue energy expenditure, and the different hypotheses involving FAO and the reactive oxygen species production in the hypothalamic control of food intake. Future Directions: The present review aims to provide an overview of novel anti-obesity strategies that target mitochondrial FAO and that will definitively be of high interest in the future research to fight against obesity-related disorders. Antioxid. Redox Signal. 00, 000000.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.