Complementarity between microhistological analysis and PCR-capillary electrophoresis in diet analysis of goats and cattle using faecal samples

Altres ajuts: project Life Montserrat (LIFE13 BIO/ES/000094). Javier Pareja was supported by the National Fund for Scientific, Technological Development and Technological Innovation (FONDECYT), the funding branch of the National Council for Science, Technology and Technological Innovation (CONCYTEC) Peru (Grant contract number N° 236-2015-FONDECYT).An evaluation is made of the complementarity between two non-invasive techniques, cuticle microhistological analysis (CMA) and PCR-capillary electrophoresis (PCR-CE) DNA-based analysis, for the determination of herbivore diet composition from faecal samples. Cuticle microhistological analysis is based on the different microanatomical characteristics of the epidermal fragments remaining in the faeces. The PCR-CE technique combines PCR amplification of a trnL(UAA) genomic DNA region with amplicon length determination by CE, with this length being characteristic for each species or taxon. A total of 37 fresh stool samples were analyzed, including 16 from feral goats (Capra hircus) from the Tramuntana mountain range (Mallorca, Baleares) and 11 from Bruna dels Pirineus cattle breed (Bos taurus) from the surrounding Montserrat mountain range (Barcelona, Spain). All the animals were in a free grazing Mediterranean pine habitat, dominated by Aleppo pine (Pinus halepensis). The results showed that both techniques detected a similar number of plant components in the faeces of goats and cows. In the case of goats, a positive correlation was obtained between the percentage of samples in which a particular taxon is detected by CMA and the percentage of samples in which that taxon is detected by PCR-CE. This correlation was not observed in the case of cows. It is concluded that PCR-CE is a fast and reliable method to detect the different plant components in the faeces of herbivores. However, it cannot be considered as an alternative to CMA, but as a complementary method, since both techniques can detect some taxa that are not detected by the other technique. In addition, CMA detected the presence of the different taxa in a greater number of samples, and at the same time, it enables quantitative data to be obtained for plant diet composition. The species of herbivore also seems to influence the results obtained by PCR-CE, so more studies are required to address this aspect

Comparing the accuracy of PCR-capillary electrophoresis and cuticle microhistological analysis for assessing diet composition in ungulates : a case study with Pyrenean chamois

The study of diet composition is required to understand the interactions between animal and plant ecosystems. Different non-invasive techniques applied on faecal samples have commonly been used for such purposes, with cuticle microhistological analysis (CMA) and emerging DNA-based methods being the most relevant. In this work, we refined and optimized a qualitative DNA-based approach combining PCR amplification of long trnL(UAA) and ITS2 fragments and capillary electrophoresis (PCR-CE), instead of short trnL(UAA) fragments and massive sequencing technologies commonly reported. To do so, we develope a controlled diet assay using a stabled Pyrenean chamois specimen (Rupicapra pirenaica pyrenaica), which included representative herbaceous and shrubby plant species. We also assessed the impact of sample freshness on the diet determination of this mountain caprinae by exposing faecal samples to the outdoor environment for three weeks. Faecal samples from both experiments were analysed by qualitative PCR-CE and semi-quantitative CMA in order to compare the pros and cons of both approaches. Our results show that all of the offered plant species were detected by both methodologies although CMA overdetected shrubs compared to herbaceous species. At the same time, sample degradation due to sustained climate exposure is a limiting factor for molecular analysis, but not for CMA. Taken all together, our results suggest that the qualitative information obtained by CMA and PCR-CE can be interchangeable when faecal samples are fresh (less than one week after deposition) but, afterwards, molecular analysis underestimates diet composition probably due to DNA degradation. CMA, however, can accurately be used at least three weeks after defecation. Moreover, by combining the results of simultaneous PCR amplification of two complementary genes, this optimized PCR-CE methodology provides a reliable, feasible and more affordable alternative for multiple and routine analyses of complex samples. Neither CMA nor PCR-CE seems to solve comprehensively the quatification of herbivore diets and thus further research needs to be done

Efecto de la simulación de ramoneo en parámetros estructurales de tres especies de matorral mallorquín

La herviboría por la cabra salvaje mallorquina y la cabra doméstica asilvestrada puede ser uno de los factores que determinen la regresión en que se encuentran las poblaciones de tres especies anteriormente abundantes y actualmente raras del matorral mallorquín: boj balear (Buxus balearica), canadillo (Ephedra fragilis) y enebro rojo (Juniperus oxycedrus). Para determinar esta afectación analizamos la respuesta de estas especies en condiciones de invernadero, a las que se les realizó una simulación de ramoneo cortando el 80% de sus brotes terminales y se les midió la variación en los parámetros estructurales de crecimiento y capacidad de generar brotes nuevos. La simulación de ramoneo produjo respuestas morfológicas diferentes en las tres especies estudiadas. Mientras en las tres especies aumenta la producción de brotes, el crecimiento en diámetro no aumenta de manera significativa en B. balerica ni en E. fragilis, y en J. oxycedrus se ve afectado negativamente. Por lo que respecta al crecimiento en altura, el ramoneo tiene un efecto positivo en B. balearica y J. oxycedrus y es indiferente en E. fragilis. El hecho de que B .balearica sea actualmente una especie poco ramoneada contribuiría a explicar su regresión

S-Nitrosoglutathione is a component of wound- and salicylic acid-induced systemic responses in Arabidopsis thaliana

S-Nitrosoglutathione (GSNO) is a bioactive, stable, and mobile reservoir of nitric oxide (NO), and an important player in defence responses to herbivory and pathogen attack in plants. It has been demonstrated previously that GSNO reductase (GSNOR) is the main enzyme responsible for the in vivo control of intracellular levels of GSNO. In this study, the role of S-nitrosothiols, in particular of GSNO, in systemic defence responses in Arabidopsis thaliana was investigated further. It was shown that GSNO levels increased rapidly and uniformly in injured Arabidopsis leaves, whereas in systemic leaves GSNO was first detected in vascular tissues and later spread over the parenchyma, suggesting that GSNO is involved in the transmission of the wound mobile signal through the vascular tissue. Moreover, GSNO accumulation was required to activate the jasmonic acid (JA)-dependent wound responses, whereas the alternative JA-independent wound-signalling pathway did not involve GSNO. Furthermore, extending previous work on the role of GSNOR in pathogenesis, it was shown that GSNO acts synergistically with salicylic acid in systemic acquired resistance activation. In conclusion, GSNOR appears to be a key regulator of systemic defence responses, in both wounding and pathogenesis

Estudis estructurals, bioquímics i funcionals de la proteïna quinasa CK2 de sistemes vegetals

Consultable des del TDXTítol obtingut de la portada digitalitzadaLa proteïna quinasa CK2 és una Ser/Thr fosfotransferasa que participa en les rutes de transducció de senyal relacionades amb la proliferació cel·lular. En la forma més freqüentment descrita, és un enzim oligomèric format per subunitat catalítica alfa i subunitat reguladora beta, que s'organitzen en un tetràmer actiu del tipus alfa2beta2. L'objectiu d'aquesta tesi doctoral fou l'estudi a nivell bioquímic i funcional de la proteïna quinasa CK2 de sistemes vegetals, especialment en relació a la proliferació cel·lular. Es va purificar per cromatografia líquida la CK2 de plantes d'Arabidopsis thaliana. En aquesta espècie, com en la majoria de les plantes, es va demostrar que coexisteixen isoformes monomèriques (alfa) amb formes oligomèriques (alfa 2beta 2), ambdues actives enzimàticament i que mostren propietats bioquímiques diferents. Per a l'estudi de la regulació de la CK2 durant la divisió cel·lular es va utilitzar com a sistema biològic la línia cel·lular BY-2 de tabac, que és altament sincronitzable. L'expressió global de les subunitats alfa i beta era constitutiva al llarg del cicle cel·lular. L'activitat enzimàtica oscil·lava al llarg del cicle i mostrava pics a la transició G1/S i a Mitosi. Es proposa que les poliamines, els moduladors al·lostèrics més importants de l'activitat CK2, podrien ser en part responsables de les oscil·lacions d'activitat. La inhibició in vivo de la CK2, amb l'inhibidor específic 4,5,6,7-tetrabromobenzotriazole, va corroborar els pics d'activitat. El bloqueig del cicle cel·lular a les fases S i G2 provocava la mort de les cèl·lules en un interval curt de temps. El bloqueig a la fase G1 no impedia que aquestes iniciessin la replicació en el moment previst, si bé no eren capaces de completar-la perquè condensaven prematurament la cromatina. Es discuteix la possible funcionalitat de la CK2 en l'acompliment del checkpoint G2/M. L'anàlisi de l'expressió i l'activitat de la CK2 a la corba de creixement de les cèl·lules BY-2, juntament amb l'establiment del patró d'expressió de la CK2 en diferents òrgans vegetals d'A. thaliana i Raphanus sativus per hibridació in situ, va revelar que aquestes augmentaven quan s'entrava a un estadi de proliferació. A més, l'activitat CK2 es regula a través de la interacció al·lostèrica amb les poliamines, així com per la presència de la subunitat beta , controlada a nivell post-transcripcional. Per hibridació in situ, es va observar una expressió coordinada de les dues subunitats, la qual era alta en tipus cel·lulars en divisió, com els meristems, els diferents primordis vegetals i el pericicle. El cribatge d'una biblioteca de cDNA de tabac va permetre aïllar dos cDNAs per a la subunitat alfa i un per a la subunitat beta, la seqüència dels quals conservava els motius estructurals característics. Postulem que la presència d'una extensió d'uns 80 aminoàcids a l'extrem N-terminal del polipèptid beta, exclusiva de les plantes, pot ser determinant pel plegament de la forma tetramèrica activa. Protein kinase CK2 is a widely distributed Ser/Thr phosphotransferase, that participates in signal transduction pathways involved in cell proliferation. The classical reported structure is that of a tetrameric enzyme, alfa 2beta 2, where _lfa is the catalytic subunit and beta is the regulatory subunit. The aim of this PhD was the study of the protein kinase CK2 in plant systems, specially in relation to cell proliferation process, at both biochemical and functional levels. Protein kinase CK2 from Arabidopsis thaliana seedlings was purified by liquid chromatography. In this plant, as it is described in other plant systems, two active isoforms coexist, one of them being monomeric (alfa) ant the other oligomeric (alfa 2beta 2), that show different biochemical properties. In order to investigate whether protein kinase CK2 is regulated during the cell division process in higher plants, the highly synchronizable tobacco BY-2 cell line was used. The expression of both alfa and beta subunits was constitutive throughout the cell cycle, while enzymatic activity showed oscillations, peacking at G1/S transition and Mitosis. The putative role of poliamines, the most important allosteric modulators of CK2, in the post-transcriptional regulation of CK2 activity during the cell cycle, is discussed. In vivo inhibition of CK2 by using the specific inhibitor 4,5,6,7-tetrabromobenzotriazole, corroborated the peaks of activity. Moreover, the blocking of the cell cycle at S and G2 phases, with the CK2 specific inhibitor, lead to the arrest of the cells in that phases and to its death shortly afterwards. In contrast, when the cells were blocked in G1, they iniciated the replication in the normal timing but they couldn't accomplish the process because the cromatin had condensed. A putative role of CK2 in the control of the G2/M checkpoint is postulated. Analysis of expression and activity of CK2 in the BY-2 growth curve, together with in situ hybridization data in different tissues in development from A. thaliana and Raphanus sativus, showed that it exists a transcriptional regulation of gene expression of both subunits when cells enter a proliferative state from a resting state. In addition, at the growth curve of the BY-2 cells, CK2 activity is regulated by controlling the _eta subunit levels by a post-transcriptional mechanism, and also by allosteric interaction with polyamines. By in situ hybridization, a coordinated expression of alfa and beta subunits in actively dividing cells, such as meristems, different organ primordia and pericicle, was observed. The screening of a tobacco cDNA library lead to the isolation of two cDNAs with homology to the alfa subunit, and one cDNA for the beta subunit. The sequences conserved the characteristic structural motifs of these proteins. The presence of an extension of aproximately 80 aminoacids at the N-terminal site of the beta subunit, which is only found in plant beta polypeptides, might be crucial for the correct folding of the active tetrameric protein

Estudio comparativo de los componentes vegetales presentes en heces de herbívoros mediante técnicas microhistológicas y moleculares

En la última década los avances en el campo de la Biología molecular han permitido el desarrollo de nuevas herramientas, como el DNA barcoding, para la identificación de las especies vegetales presentes en muestras complejas con ADN altamente degradado, como es el caso de las heces de herbívoros. El DNA barcoding consiste en la amplificación de un gen marcador específico, por ejemplo el gen cloroplástico trnL(UAA), mediane PCR (Reacción en Cadena de Polimerasa), seguida de la determinación de la longintud de los amplicones o, alternativamente, de secuenciación masiva y comparación de las secuencias obtenidas con aquellas presentes en los bancos de datos. Hemos realizado un estiduo comparativo de la composición de la dieta de la Cabra Salvaje Mallorquina que habita en matorrales mediterráneos, empleando dos técnicas: el análisis microhistológico y el DNA barcoding. Nuestros resultados preliminares muestran que los análisis moleculares aportan resultados robustos, reproducibles, rápidos de obtener y a un coste económico asumible. Además, las dos metodologías utilizadas podrían ser complementarias y proporcionarían datos concluyentes sobre la composición de la dieta de los animales hasta el nivel taxonómico de especie

S-Nitrosoglutathione Reductase Affords Protection against Pathogens in Arabidopsis, Both Locally and Systemically

Nitric oxide and S-nitrosothiols (SNOs) are widespread signaling molecules that regulate immunity in animals and plants. Levels of SNOs in vivo are controlled by nitric oxide synthesis (which in plants is achieved by different routes) and by S-nitrosoglutathione turnover, which is mainly performed by the S-nitrosoglutathione reductase (GSNOR). GSNOR is encoded by a single-copy gene in Arabidopsis (Arabidopsis thaliana; Martínez et al., 1996; Sakamoto et al., 2002). We report here that transgenic plants with decreased amounts of GSNOR (using antisense strategy) show enhanced basal resistance against Peronospora parasitica Noco2 (oomycete), which correlates with higher levels of intracellular SNOs and constitutive activation of the pathogenesis-related gene, PR-1. Moreover, systemic acquired resistance is impaired in plants overexpressing GSNOR and enhanced in the antisense plants, and this correlates with changes in the SNO content both in local and systemic leaves. We also show that GSNOR is localized in the phloem and, thus, could regulate systemic acquired resistance signal transport through the vascular system. Our data corroborate the data from other authors that GSNOR controls SNO in vivo levels, and shows that SNO content positively influences plant basal resistance and resistance-gene-mediated resistance as well. These data highlight GSNOR as an important and widely utilized component of resistance protein signaling networks conserved in animals and plants