An elicitor isolated from smut teliospores (Sporisorium scitamineum) enhances lignin deposition on the cell wall on both sclerenchyma and xylem in sugarcane leaves

Sugarcane leaf shows the classical arrangement of cells which defines a C4 species. Vascular bundles consist of xylem, phloem and fibres, surrounded by an outer layer of sclereids and an inner ring of stone cells associated with the phloem. Some sclereids located below and above the vascular bundles act as docking cells and connect the vascular bundle to the internal surfaces of upper and lower layers of the epidermis. A compact mass of sclereids occupies the total internal volume of the leaf edge. Neither docking cells nor the internal mass of sclereids in the edge were markedly coloured by acriflavin or phloroglucinol, indicating the absence of lignin in their cell walls. However, such staining indicated that fibres of the vascular bundle and the external layer of sclereids were strongly lignified. Incubation of leaf discs with an elicitor produced by the pathogen Sporisorium scitamineum increased the thickness of the lignified cell walls of sclereids as well as the mid and small xylem vessels, as a possible mechanical defense response to the potential entry of the pathogen.

Arabidopsis heterotrimeric G-protein regulates cell wall defense and resistance to necrotrophic fungi

The Arabidopsis heterotrimeric G-protein controls defense responses to necrotrophic and vascular fungi. The agb1 mutant impaired in the Gβ subunit displays enhanced susceptibility to these pathogens. Gβ/AGB1 forms an obligate dimer with either one of the Arabidopsis Gγ subunits (γ1/AGG1 and γ2/AGG2). Accordingly, we now demonstrate that the agg1 agg2 double mutant is as susceptible as agb1 plants to the necrotrophic fungus Plectosphaerella cucumerina. To elucidate the molecular basis of heterotrimeric G-protein-mediated resistance, we performed a comparative transcriptomic analysis of agb1-1 mutant and wild-type plants upon inoculation with P. cucumerina. This analysis, together with metabolomic studies, demonstrated that G-protein-mediated resistance was independent of defensive pathways required for resistance to necrotrophic fungi, such as the salicylic acid, jasmonic acid, ethylene, abscisic acid, and tryptophan-derived metabolites signaling, as these pathways were not impaired in agb1 and agg1 agg2 mutants. Notably, many mis-regulated genes in agb1 plants were related with cell wall functions, which was also the case in agg1 agg2 mutant. Biochemical analyses and Fourier Transform InfraRed (FTIR) spectroscopy of cell walls from G-protein mutants revealed that the xylose content was lower in agb1 and agg1 agg2 mutants than in wild-type plants, and that mutant walls had similar FTIR spectratypes, which differed from that of wild-type plants. The data presented here suggest a canonical functionality of the Gβ and Gγ1/γ2 subunits in the control of Arabidopsis immune responses and the regulation of cell wall composition.

Xyloglucan endotransglucosylase and cell wall extensibility

Transgenic tomato hypocotyls with altered levels of an XTH gene were used to study how XET activity could affect the hypocotyl growth and cell wall extensibility. Transgenic hypocotyls showed significant over-expression (line 13) or co-suppression (line 33) of the SlXTH1 in comparison with the wild type, with these results being correlated with the results on specific soluble XET activity, suggesting that SlXTH1 translates mainly for a soluble XET isoenzyme. A relationship between XET activity and cell wall extensibility was found, and the highest total extensibility was located in the apical hypocotyl segment of the over-expressing SlXTH1 line, where the XET-specific activity and hypocotyl growth were also highest compared with the wild line.

Xyloglucan endotransglucosylase/hydrolase (XTH) overexpression affects growth and cell wall mechanics in etiolated Arabidopsis hypocotyls.

Growth and biomechanics of etiolated hypocotyls from Arabidopsis thaliana lines overexpressing xyloglucan endotransglucosylase/hydrolase AtXTH18, AtXTH19, AtXTH20, and PttXET16-34 were studied. Overexpression of AtXTH18, AtXTH19, and AtXTH20 stimulated growth of hypocotyls, while PttXET16-34 overexpression did not show this effect. In vitro extension of frozen/thawed hypocotyls measured by a constant-load extensiometer started from a high-amplitude initial deformation followed by a slow time-dependent creep. Creep of growing XTH-overexpressing (OE) hypocotyls was more linear in time compared with the wild type at pH 5.0, reflecting their higher potential for long-term extension. XTH-OE plants deposited 65?84% more cell wall material per hypocotyl cross-sectional area than wild-type plants. As a result, their wall stress under each external load was lower than in the wild-type. Growing XTH-OE hypocotyls had higher values of initial deformation·stress?1 compared with the wild type. Plotting creep rates for each line under different loads against the respective wall stress values gave straight lines. Their slopes and intercepts with the abscissa correspond to ? (in vitro cell wall extensibility) and y (in vitro cell wall yield threshold) values characterizing cell wall material properties. The wall material in XTH-OE lines was more pliant than in the wild type due to lower y values. In contrast, the acid-induced wall extension in vitro resulted from increasing ? values. Thus, three factors contributed to the XTH-OE-stimulated growth in Arabidopsis hypocotyls: their more linear creep, higher values of initial deformation·stress?1, and lower y values.

Chemical and structural analysis of Eucalyptus globulus and E. camaldulensis leaf cuticles: a lipidized cell wall region

The plant cuticle has traditionally been conceived as an independent hydrophobic layer that covers the external epidermal cell wall. Due to its complexity, the existing relationship between cuticle chemical composition and ultra-structure remains unclear to date. This study aimed to examine the link between chemical composition and structure of isolated, adaxial leaf cuticles of Eucalyptus camaldulensis and E. globulus by the gradual extraction and identification of lipid constituents (cutin and soluble lipids), coupled to spectroscopic and microscopic analyses. The soluble compounds and cutin monomers identified could not be assigned to a concrete internal cuticle ultra-structure. After cutin depolymerization, a cellulose network resembling the cell wall was observed, with different structural patterns in the regions ascribed to the cuticle proper and cuticular layer, respectively. Our results suggest that the current cuticle model should be revised, stressing the presence and major role of cell wall polysaccharides. It is concluded that the cuticle may be interpreted as a modified cell wall region which contains additional lipids. The major heterogeneity of the plant cuticle makes it difficult to establish a direct link between cuticle chemistry and structure with the existing methodologies.

The role of the secondary cell wall in plant resistance to pathogens

Plant resistance to pathogens relies on a complex network of constitutive and inducible defensive barriers. The plant cell wall is one of the barriers that pathogens need to overcome to successfully colonize plant tissues. The traditional view of the plant cell wall as a passive barrier has evolved to a concept that considers the wall as a dynamic structure that regulates both constitutive and inducible defense mechanisms, and as a source of signaling molecules that trigger immune responses. The secondary cell walls of plants also represent a carbon-neutral feedstock (lignocellulosic biomass) for the production of biofuels and biomaterials. Therefore, engineering plants with improved secondary cell wall characteristics is an interesting strategy to ease the processing of lignocellulosic biomass in the biorefinery. However, modification of the integrity of the cell wall by impairment of proteins required for its biosynthesis or remodeling may impact the plants resistance to pathogens. This review summarizes our understanding of the role of the plant cell wall in pathogen resistance with a focus on the contribution of lignin to this biological process.

Genes encoding beta-mannanases in Brachypodium distachyon seeds have a role not only in cell wall softening upon germination sensu stricto but also during post-germinative reserve mobilization

En la coleorriza en semillas de Brachypodium son abundantes los mananos y estos van desapareciendo conforme progresa la germinación (entre 12-27 h), al mismo tiempo se observa un pico de actividad endo-beta-mananasa. Se ha establecido que de los 6 miembros de la familia MAN en B. distachyon 3 se expresan en el embrión en germinación y BdMAN3 también es abundante en la aleurona.

Signwalling: signals derived from arabiopsis cell wall activate specific resistance to pathogens

The cell wall is a dynamic structure that regulates both constitutive and inducible plant defence responses. Different molecules o DAMPs (damage-associated molecular patterns) can be released from plant cell walls upon pathogen infection or wounding and can trigger immune responses. To further characterize the function of cell wall on the regulation of these immune responses, we have performed a biased resistance screening of putative/well-characterized primary/secondary Arabidopsis thaliana cell wall mutants (cwm). In this screening we have identified more than 20 cwm mutants with altered susceptibility/resistance to at least one of the following pathogens: the necrotrophic fungi Plectosphaerella cucumerina, the vascular bacterium Ralstonia solanacearum, the biotrophic oomycete Hyaloperonospora arabidopsidis and the powdery mildew fungus Erisyphe cruciferarum. We found that cell wall extracts from some of these cwm plants contain novel DAMPs that activate immune responses and conferred enhanced resistance to particular pathogens when they were applied to wild-type plants. Using glycomic profiling we have performed an initial characterization of the active carbohydrate structures present in these cwm wall fractions, and we have determined the signalling pathways regulated by thesse fractions. . The data generated with this collection of wall mutants support the existence of specific correlations between cell wall structure/composition, resistance to particular type of pathogens and plant fitness. Remarkably, we have identified specific cwm mutations that uncoupled resistance to pathogens from plant trade-offs, further indicating the plasticity of wall structures in the regulation of plant immune responses.

Co-aggregation ability of cell Wall components of Saccharomyces cerevisiae to pathogenic bacteria

Autoaggregation in bacteria is the phenomenon of aggregation between cells of the same strain, whereas coaggregation is due to aggregation occurring among different species. Aggregation ability of prebiotic bacteria is related to adhesion ability, which is a prerequisite for the colonization and protection of the gastrointestinal tract in all animal species; however, coaggregation ability of prebiotic bacteria offers a possibility of close interaction with pathogenic bacteria.

Compatibilidad de Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) y Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae), importantes enemigos naturales de la mosca blanca Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) en cultivos hortícolas, con nuevas barreras físicas selectivas y modernos plaguicidas

Los programas de Gestión Integrada de Plagas (GIP) promueven el uso de estrategias de control que sean respetuosas con el medio ambiente, sin embargo el uso de insecticidas en los cultivos hortícolas sigue siendo necesario para el control de determinadas plagas, como es el caso de la mosca blanca Bemisia tabaci (Gennadius). Por ello, el objetivo de esta tesis es el estudio de la integración de las tres estrategias de control más empleadas hoy en día para el control de plagas: el control biológico, el físico y el químico. Una primera parte de este trabajo ha consistido en el estudio de los efectos letales y subletales de once insecticidas, aplicados a la dosis máxima de campo, sobre los enemigos naturales Eretmocerus mundus Mercet y Amblyseius swirskii Athias-Henriot, mediante ensayos de laboratorio y persistencia (laboratorio extendido). Para la evaluación de la toxicidad de los insecticidas sobre los estados de vida más protegidos de estos enemigos naturales, se trataron bajo la Torre de Potter las pupas de E. mundus y los huevos de A. swirskii. Además, se llevaron a cabo ensayos de contacto residual para determinar los efectos letales y subletales de estos insecticidas sobre el estado adulto de ambas especies de enemigos naturales. Para ello, los pesticidas se aplicaron sobre placas de cristal (laboratorio) o sobre plantas (laboratorio extendido: persistencia). Los resultados mostraron que los insecticidas flonicamida, flubendiamida, metaflumizona, metoxifenocida, spiromesifen y spirotetramat eran compatibles con el estado de pupa de E. mundus (OILB 1: Inocuos). Sin embargo, abamectina, deltametrina y emamectina fueron categorizadas como ligeramente tóxicas (OILB 2) al causar efectos deletéreos. Los dos pesticidas más tóxicos fueron spinosad y sulfoxaflor, los cuales redujeron significativamente la emergencia de las pupas tratadas (OILB 4: Tóxicos). Flonicamida, flubendiamida, metoxifenocida y spiromesifen fueron compatibles con el estado adulto de E. mundus (OILB 1: Inocuos). Abamectina, deltametrina, emamectina, metaflumizona y spiromesifen pueden ser recomendados para su uso en programas de GIP, si se usan los plazos de seguridad apropiados, de acuerdo con la persistencia de cada uno de estos insecticidas, antes de la liberación del enemigo natural. Al contrario, spinosad y sulfoxaflor no resultaron ser compatibles (OILB D: Persistentes), aunque la realización de ensayos adicionales es necesaria para ver los efectos de los mismos en campo. Todos los insecticidas estudiados, excepto el spirotetramat (OILB 2: Ligeramente tóxico), fueron selectivos para el estado de huevo de A. swirskii (OILB 1: Inocuos). Flonicamida, flubendiamida, metaflumizona, metoxifenocida, spiromesifen, spirotetramat y sulfoxaflor, fueron compatibles con el estado adulto de A. swirskii (OILB 1: Inocuos). Abamectina, deltametrina, emamectina y spinosad pueden ser recomendados para su uso en programas de GIP, si se usan los plazos de seguridad apropiados, de acuerdo con la persistencia de cada uno de estos insecticidas, antes de la liberación del enemigo natural. Entre las nuevas estrategias de la GIP, los plásticos y mallas fotoselectivas han demostrado ser una herramienta importante para el control de plagas y enfermedades en cultivos hortícolas protegidos. Por ello, en una segunda parte de este trabajo, se estudiaron tanto los efectos directos, como la combinación de efectos directos y mediados por planta y plaga de ambientes pobres en luz UV, en presencia o ausencia del Virus del rizado amarillo del tomate (TYLCV), sobre E. mundus. En primer lugar, se realizó un ensayo al aire libre para la evaluación de la capacidad de vuelo de E. mundus en cajas tipo túnel (1 x 0,6 x 0,6 m) cubiertas con distintas barreras absorbentes de luz UV. Se detectó un efecto directo en la capacidad de orientación de E. mundus, debido a que este parasitoide utiliza estímulos visuales para localizar a sus huéspedes, únicamente en las barreras que bloqueaban más del 65% de la luz UV (malla G). En segundo lugar, bajo condiciones de invernadero, se evaluó la combinación de efectos directos y mediados por planta y plaga sobre E. mundus, usando plantas de tomate sanas o infectadas con el TYLCV y cajas (30 x 30 x 60 cm) cubiertas con los distintos plásticos fotoselectivos. En este caso, no se observó ningún efecto en la capacidad benéfica del parasitoide cuando este estaba en contacto con plantas de tomate infestadas con ninfas de B. tabaci, lo que demuestra que este insecto usa estímulos táctiles para encontrar a sus huéspedes a cortas distancias. Además, las diferentes condiciones de radiación UV estudiadas tuvieron cierto impacto en la morfología, fisiología y bioquímica de las plantas de tomate, infestadas o no con el virus de la cuchara, detectándose pequeñas alteraciones en alguno de los parámetros estudiados, como el peso fresco y seco, el contenido en H y el espesor de las cutículas y de las paredes celulares de la epidermis foliar. Por último, no se observaron efectos de la radiación UV mediados por planta, ni en B. tabaci ni en su parasitoide, E. mundus. En una tercera parte, se evaluaron los efectos de una malla tratada con bifentrin sobre ambos enemigos naturales, en ensayos de laboratorio, semicampo y campo. Las mallas tratadas fueron diseñadas originariamente para el control de mosquitos vectores de la malaria, y actualmente se está trabajando para su uso en agricultura, como una nueva estrategia de control de plagas. En ensayos de laboratorio, cuando adultos de E. mundus y A. swirskii se expusieron por contacto durante 72 horas con la malla tratada (cajas de 6 cm diámetro), se registró una alta mortalidad. Sin embargo, en el ensayo de preferencia, estos enemigos naturales no fueron capaces de detectar la presencia de bifentrin y, en aquellos individuos forzados a atravesar la malla tratada, no se observó mortalidad a corto plazo (72 horas). En estudios de semicampo, llevados a cabo bajo condiciones de invernadero en cajas de 25 x 25 x 60 cm de altura, la capacidad benéfica de E. mundus no se vio afectada. Finalmente, en ensayos de campo llevados a cabo en invernaderos comerciales (4000m2) en Almería, A. swirskii no se vio afectado por la presencia en el cultivo de la malla tratada con bifentrin y los niveles de infestación de B. tabaci y F. occidentalis detectados bajo dicha malla, fueron inferiores a los del control. Por último, se ha evaluado la composición de la microflora bacteriana de tres especies de parasitoides, E. mundus, Eretmocerus eremicus Rose & Zolnerowich y Encarsia formosa Gahan, y la influencia de la misma en su susceptibilidad a insecticidas. Se llevó a cabo una extracción total de ADN de los insectos y la región variable V4 del ARNr se amplificó usando cebadores universales bacterianos. Para identificar las secuencias de los géneros bacterianos presentes en los parasitoides, se realizó una Next Generation sequencing (Illumina sequencing). Una vez identificados los géneros bacterianos, el gen ADNr 16S de las Actinobacterias se amplificó del ADN extraído de los insectos, usando cebadores universales bacterianos y específicos de Actinobacterias, y los productos de la Nested PCR fueron clonados para identificar todas las especies del género Arthrobacter. Tres bacterias (A. aurescens Phillips, A. nicotinovarans Kodama, Yamamoto, Amano and Amichi y A. uratoxydans Stackebrandt, Fowler, Fiedler and Seiler), próximas a las especies de Arthrobacter presentes en los parasitoides, se obtuvieron de la colección bacteriana del BCCMTM/LMG y se midió su actividad esterasa. Finalmente, se realizaron ensayos con antibióticos (tetraciclina) y de contacto residual con insecticidas (abamectina) para determinar la influencia de las especies de Arthrobacter en la susceptibilidad de E. mundus a insecticidas. Los resultados muestran que este género bacteriano puede afectar a la toxicidad de E. mundus a abamectina, mostrando la importancia de la comunidad microbiana en enemigos naturales, factor que debe ser considerado en los estudios de evaluación de los riesgos de los insecticidas. ABSTRACT Integrated Pest Management (IPM) programs promote the use of control strategies more respectful with the environment; however the use of insecticides in vegetable crops is still needed to control certain pests, such as the whitefly Bemisia tabaci (Gennadius). Therefore, the objective of this work is to study the integration of the three most commonly used pest control strategies nowadays: biological, physical and chemical control. Firstly, the lethal and sublethal effects of eleven insecticides, applied at their maximum field recommended concentration, on the parasitic wasp Eretmocerus mundus Mercet and the predator Amblyseius swirskii Athias-Henriot has been assessed in the laboratory and in persistence tests (extended laboratory). To test the effects of pesticides on the most protected life stage of these natural enemies, E. mundus pupae and A. swirskii eggs were sprayed under a Potter precision spray tower. Laboratory contact tests were therefore conducted to determine the lethal and sublethal effects of these pesticides on the adult stage of these natural enemies. In the residual contact tests the pesticides were applied on glass plates (laboratory) or plants (extended laboratory: persistence). The study showed that the insecticides flonicamid, flubendiamide, metaflumizone, methoxyfenozide, spiromesifen and spirotetramat were selective for E. mundus pupae (IOBC 1: Harmless). Nevertheless, abamectin, deltamethrin and emamectin were categorized as slightly harmful (IOBC 2) due to the deleterious effects caused. The two most harmful pesticides were spinosad and sulfoxaflor, which significantly reduced the adult emergence from treated pupae (IOBC 4: Harmful). Flonicamid, flubendiamide, methoxyfenozide and spiromesifen were compatible with E. mundus adults (IOBC 1: Harmless). Base on the duration of the harmful activity, abamectin, deltamethrin, emamectin, metaflumizone and spirotetramat could be recommended for use in IPM programs if appropriate safety deadlines are used before the natural enemy release. On the contrary, spinosad and sulfoxaflor were not compatible (IOBC D: persistent), although additional studies are required to determine their effects under field conditions. All the pesticides tested, except spirotetramat (IOBC 2: Slightly harmful), were selective for A. swirskii eggs (IOBC 1: Harmless). Flonicamid, flubendiamide, metaflumizone, methoxyfenozide, spiromesifen, spirotetramat and sulfoxaflor were compatible with A. swirskii adults (IOBC 1: Harmless). However, abamectin, deltamethrin, emamectin and spinosad could be recommended for use in IPM programs if appropriate safety deadlines are used before the natural enemy release. Among new IPM strategies, UV-absorbing photoselective plastic films and nets have been shown to be an important tool for the control of pests and diseases in horticultural protected crops. Because of that, we secondly studied the plant and pest insect-mediated and/or the direct effects on E. mundus under different UV radiation conditions, in presence or absence of the Tomato Yellow Leaf Curl Virus (TYLCV). In the first experiment, performed outdoors, the flight activity of E. mundus was studied in one-chamber tunnels (1 x 0.6 x 0.6 m) covered with different photoselective barriers. Because E. mundus uses visual cues for host location at a long distance, a direct effect on its host location ability was detected, but only in the UV-absorbing barriers blocking more than 65% of the UV light (G net). In a second experiment, the direct and plant and pest insect-mediated effects of different UV radiation conditions on E. mundus were studied, inside cages (30 x 30 x 60 cm) covered with the different UVplastic films and under greenhouse conditions, using healthy or TYLCV-virus infected tomato plants. In this case, not any effect on the beneficial capacity of this parasitoid was detected, proving that he uses tactile cues at a short distance of the host. Moreover, the different UV radiation conditions studied had a certain direct impact in the morphology, physiology and biochemistry of tomato plants infested or not with the TYLCV, and small alterations in some parameters such as fresh and dry weight, H percentage and cuticle and cell wall thickness of epidermal cells of the leaves, were detected. Finally, none plant-mediated UV effects neither in the whitefly B. tabaci nor in their parasitic wasp were found. Thirdly, the effects of a bifenthrin treated net were evaluated in different laboratory, semi-field and field experiments on the natural enemies studied. Treated nets were developed long time ago aiming at the control of the mosquitoes vectors of malaria, and nowadays, there is a great interest on assessing the possibility of their use in agriculture. In laboratory assays, a high mortality was recorded when E. mundus and A. swirskii adults were exposed by contact to the bifenthrin treated net for 72 hours in small cages (12 cm diameter). However, these natural enemies were not able to detect the presence of bifenthrin in a dual-choice test and no short-term mortality (72 hours) was recorded in those individuals that went through the treated net. In semi-field assays, performed under greenhouse conditions with cages of 25 x 25 x 60 cm high, the beneficial capacity of E. mundus was not affected. Finally, in field assays carried out in commercial multispan greenhouses (4000 m2) in Almería, A. swirskii was not affected by the presence of the bifenthrin treated net in the crop and the B. tabaci and F. occidentalis infestation levels were significantly lower than in the control. Finally, the composition of the microflora present in three species of parasitoids, E. mundus, Eretmocerus eremicus Rose & Zolnerowich and Encarsia formosa Gahan, and its influence in their susceptibility to insecticides, have been assessed. A total DNA extraction was performed on insects and universal bacterial primers were used to amplify the variable V4 region of the rRNA. A Next Generation sequencing (Illumina sequencing) was performed to identify the sequences of the bacterial genera present in the parasitic wasps. Once, the bacterial genera were identified, 16S rDNA gene of Actinobacteria were amplified from insects DNA extracts using the universal bacterial and actinobacterial primers, and the nested PCR products, were cloned to identify the Arthrobacter species. Three bacteria (A. aurescens Phillips, A. nicotinovarans Kodama, Yamamoto, Amano and Amichi and A. uratoxydans Stackebrandt, Fowler, Fiedler and Seiler), having the closest match with the Arthrobacter species present in the parasitic wasps, were obtained from the BCCMTM/LMG bacteria collection and its esterase activity was measured. Finally, antibiotic and residual contact tests were done to determine the influence of Arthrobacter species in the susceptibility of E. mundus to pesticides (abamectin). The results suggest that this bacterial genus can affect the toxicity of E. mundus to abamectin, which in turn supports the importance of the microbial community in natural enemies that it should be considered as a factor in risk assessment tests of pesticides.

The Agrobacterium vitis T-6b oncoprotein induces auxin-independent cell expansion in tobacco

Among the Agrobacterium T-DNA genes, rolB, rolC, orf13, orf8, lso, 6b and several other genes encode weakly homologous proteins with remarkable effects on plant growth. The 6b oncogene induces tumors and enations. In order to study its properties we have used transgenic tobacco plants that carry a dexamethasone-inducible 6b gene, dex-T-6b. Upon induction, dex-T-6b plants develop a large array of morphological modifications, some of which involve abnormal cell expansion. In the present investigation, dex-T-6b-induced expansion was studied in intact leaves and an in vitro leaf disc system. Although T-6b and indole-3-acetic acid (IAA) both induced expansion and were non-additive, T-6b expression did not increase IAA levels, nor did it induce an IAA-responsive gene. Fusicoccin (FC) is known to stimulate expansion by increasing cell wall plasticity. T-6b- and FC-induced expansion were additive at saturating FC concentrations, indicating that T-6b does not act by a similar mechanism to FC. T-6b expression led to higher leaf osmolality values, in contrast to FC, suggesting that the T-6b gene induces expansion by increasing osmolyte concentrations. Metabolite profiling showed that glucose and fructose played a major role in this increase. We infer that T-6b disrupts the osmoregulatory controls that govern cell expansion during development and wound healing.

Softening-up mannan-rich cell walls

The softening and degradation of the cell wall (CW), often mannan enriched, is involved in several processes during development of higher plants, such as meristematic growth, fruit ripening, programmed cell death, and endosperm rupture upon germination. Mannans are also the predominant hemicellulosic CW polymers in many genera of green algae. The endosperm CWs of dry seeds often contain mannan polymers, sometimes in the form of galactomannans (Gal-mannans). The endo-beta-mannanases (MANs) that catalyse the random hydrolysis of the beta-linkage in the mannan backbone are one of the main hydrolytic enzymes involved in the loosening and remodelling of CWs. In germinating seeds, the softening of the endosperm seed CWs facilitates the emergence of the elongating radicle. Hydrolysis and mobilization of endosperm Gal-mannans by MANs also provides a source of nutrients for early seedling growth, since Gal-mannan, besides its structural role, serves as a storage polysaccharide. Therefore, the role of mannans and of their hydrolytic enzymes is decisive in the life cycle of seeds. This review updates and discusses the significance of mannans and MANs in seeds and explores the increasing biotechnological potential of MAN enzymes.

Distinct and conserved transcriptomic changes during nematode-induced giant cell development in tomato compared with Arabidopsis: a functional role for gene repression

Root-knot nematodes (RKNs) induce giant cells (GCs) from root vascular cells inside the galls. Accompanying molecular changes as a function of infection time and across different species, and their functional impact, are still poorly understood. Thus, the transcriptomes of tomato galls and laser capture microdissected (LCM) GCs over the course of parasitism were compared with those of Arabidopsis, and functional analysis of a repressed gene was performed. Microarray hybridization with RNA from galls and LCM GCs, infection-reproduction tests and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) transcriptional profiles in susceptible and resistant (Mi-1) lines were performed in tomato. Tomato GC-induced genes include some possibly contributing to the epigenetic control of GC identity. GC-repressed genes are conserved between tomato and Arabidopsis, notably those involved in lignin deposition. However, genes related to the regulation of gene expression diverge, suggesting that diverse transcriptional regulators mediate common responses leading to GC formation in different plant species. TPX1, a cell wall peroxidase specifically involved in lignification, was strongly repressed in GCs/galls, but induced in a nearly isogenic Mi-1 resistant line on nematode infection. TPX1 overexpression in susceptible plants hindered nematode reproduction and GC expansion. Time-course and cross-species comparisons of gall and GC transcriptomes provide novel insights pointing to the relevance of gene repression during RKN establishment.

Nutritional impact on health and performance in intensively reared rabbits

The present work summarizes research related to the definition of nutrient recommendations for feeds used in the intensive production of rabbit's meat. Fibre is the main chemical constituent of rabbit diets that typically contain 320 to 360 and 50 to 90 g/kg of insoluble and soluble fibre, respectively. Instead, the dietary contents of cereal grains (∼120 to 160 g/kg), fat (15 to 25 g/kg) and protein concentrates (150 to 180 g/kg) are usually low with respect to other intensively reared monogastric animals. Cell wall constituents are not well digested in rabbits, but this effect is compensated by its stimulus of gut motility, which leads to an increasing rate of passage of digesta, and allows achieving an elevated dry matter intake. A high feed consumption and an adequate balance in essential nutrients are required to sustain the elevated needs of high-productive rabbits measured either as reproductive yield, milk production or growth rate in the fattening period. Around weaning, pathologies occur in a context of incomplete development of the digestive physiology of young rabbits. The supply of balanced diets has also been related to the prevention of disorders by means of three mechanisms: (i) promoting a lower retention time of the digesta in the digestive tract through feeding fibre sources with optimal chemical and physical characteristics, (ii) restricting feed intake after weaning or (iii) causing a lower flow of easily available substrates into the fermentative area by modifying feed composition (e.g. by lowering protein and starch contents, increasing its digestibility or partially substituting insoluble with soluble fibre), or by delaying age at weaning. The alteration in the gut microbiota composition has been postulated as the possible primary cause of these pathologies.

Identificación y caracterización de la ruta mediada por la proteína G heterotrimérica de Arabidopsis thaliana en la respuesta inmune

La resistencia de las plantas a los hongos necrótrofos como Plectosphaerella cucumerina es genéticamente compleja y depende de la activación coordinada de distintas rutas de señalización (Llorente et al, 2005; Sanchez-Vallet et al, 2010). Entre éstas se encuentran las mediadas por la proteína G heterotrimérica, un complejo formado por tres subunidades (Gα, Gβ y Gγ) que regula tanto la respuesta de inmunidad a diferentes patógenos como distintos procesos de desarrollo (Temple and Jones, 2007). En esta Tesis hemos demostrado que, en Arabidopsis, el monómero funcional formado por las subunidades Gβ y Gγ1/Gγ2 es el responsable de la regulación de la respuesta de defensa, ya que mutantes nulos en estas subunidades (agb1 y agg1 agg2) presentan una alta susceptibilidad al hongo P. cucumerina. Además, hemos identificado varios aminoácidos (Q102, T188 y R235) de la proteína AGB1 esenciales en la interacción con los efectores correspondientes para la regulación de la respuesta inmune (Jiang et al, enviado). Para determinar las bases moleculares de la resistencia mediada por la proteína G heterotrimérica, llevamos a cabo un análisis transcriptómico comparativo entre los genotipos agb1 y Col-0, el cual reveló que la resistencia mediada por AGB1 no depende de rutas defensivas implicadas en la resistencia a hongos necrotrofos, como las mediadas por el ácido salicílico (SA), etileno (ET), jasmónico (JA) o ácido abscísico (ABA), o la ruta de biosíntesis de metabolitos derivados del triptófano. Este estudio mostró que un número significativo de los genes desregulados en respuesta a P. cucumerina en el genotipo agb1 respecto a las plantas silvestres codificaban proteínas con funciones relacionadas con la pared celular. La evaluación de la composición y estructura de la pared de los mutantes de las subunidades de la proteína G heterotrimérica reveló que los genotipos agb1 y agg1 agg2 presentaban alteraciones similares diferentes de las observadas en plantas silvestres Col-0, como una reducción significativa en el contenido de xilosa en la pared. Estos datos sugieren que la proteína G heterotrimérica puede modular la composición/estructura de la pared celular y contribuir, de esta manera, en la regulación de la respuesta inmune (Delgado- Cerezo et al, 2011). La caracterización del interactoma de la proteína G heterotrimérica corroboró la relevancia funcional que presenta en la regulación de la pared celular, ya que un número significativo de las interacciones identificadas estaban comprendidas por proteínas relacionadas directa o indirectamente con la biogénesis y remodelación de la pared celular (Klopffleisch et al, 2011). El papel en inmunidad de algunos de estos potenciales efectores ha sido validado mediante el análisis de la resistencia a P. cucumerina de los mutantes de pérdida de función correspondientes. Con el objetivo de caracterizar las rutas de señalización mediadas por AGB1 e identificar efectores implicados en esta señalización, llevamos a cabo una búsqueda de mutantes supresores de la susceptibilidad de agb1 a P. cucumerina, identificándose varios mutantes sgb (supressor of Gbeta). En esta Tesis hemos caracterizado en detalle el mutante sgb10, que presenta una activación constitutiva de las rutas de señalización mediadas por SA y JA+ET y suprime el fenotipo de susceptibilidad de agb1. SGB10 y AGB1 forman parte de rutas independientes en la regulación de la respuesta inmune, mientras que interaccionan de forma compleja en el control de determinados procesos de desarrollo. La mutación sgb10 ha sido cartografiada entre los genes At3g55010 y At3g56408, que incluye una región con 160 genes. ABSTRACT Plant resistance to necrotrophic fungi Plectosphaerella cucumerina is genetically complex and depends on the interplay of different signalling pathways (Llorente et al, 2005; Sanchez-Vallet et al, 2010). Among others, the heterotrimeric G protein complex has a relevant role. The G protein that is formed by three subunits (Gα, Gβ and Gγ) is a pleiotropic regulator of immune responses to different types of pathogens and developmental issues (Temple and Jones, 2007). Throughout the Thesis, we have demonstrated that Arabidopsis’ functional monomer formed by the Gβ and Gγ1/Gγ2 subunits is a key regulator of defense response, as null mutants (agb1 and agg1 agg2) are equally hypersusceptible to P. cucumerina infection. In addition we have identified several AGB1 aminoacids (Q102, T188 y R235) essentials to interact with specific effectors during the regulation of immune response (Jiang et al, sent).To determine the molecular basis of heterotrimeric G protein mediated resistance we have performed a microarray analysis with agb1-1 and wild type Col-0 plants before and after P. cucumerina challenge. A deep and exhaustive comparative transcriptomical analysis of these plants revealed that AGB1 mediated resistance does not rely on salicilic acid (SA), ethylene (ET), jasmonates (JA), abscisic acid (ABA) or triptophan derived metabolites biosynthesis. However the analysis revealed that a significant number of cell wall related genes are misregulated in the agb1 mutant after pathogen challenge when compared to wild-type plants. The analysis of cell wall composition and structure showed similar cell wall alterations between agb1 and agg1 agg2 mutants that are different from those of wild-type plants, so far the mutants present a significant reduction in xylose levels. All these results suggest that heterotrimeric G protein may regulate immune response through modifications in the cell wall composition/structure (Delgado-Cerezo et al, 2011). The characterization of Heterotrimeric G protein interactome revealed highly connected interactions between the G-protein core and proteins involved in cell wall composition or structure (Klopffleisch et al, 2011). To test the role in immunity of several effectors identified above, we have performed resistance analysis of corresponding null mutants against P. cucumerina. In order to characterize AGB1 mediated signalling pathway and identify additional effectors involved in AGB1-mediated immune response against P. cucumerina, we have performed a screening to isolate mutants with suppression of agb1 phenotype. One of the mutants, named sgb10, has been characterized during the Thesis. The mutant shows constitutive expression of SA, JA+ET-mediated defense signaling pathways to suppres agb1 hypersusceptibility. SGB10 and AGB1 proteins seem to be part of independent pathways in immunity, however its function during development remains unclear. At present, we have mapped the sgb10 mutation between At3g55010 and At3g56408 genes. This region contains 160 genes.