Heterologous Expression of a Plant Small Heat-Shock Protein Enhances Escherichia Coli Viability under Heat And Cold Stress Ref.: 1

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17.5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37°C to 50°C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4°C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress

Chaperonas moleculares y tolerancia a estrés abiótico en especies arbóreas

Las temperaturas extremas, la sequía y otros estreses abióticos limitan la producción forestal de forma significativa, causando grandes pérdidas económicas en el sector. Los árboles, al ser organismos sésiles, han desarrollado una serie de estrategias para percibir dichos factores, activando respuestas defensivas apropiadas. Entre ellas ocupa un lugar preeminente la síntesis de proteínas con actividad chaperona molecular. Las chaperonas moleculares interaccionan con proteínas desnaturalizadas total o parcialmente, promoviendo su correcto plegamiento y ensamblaje. Las chaperonas moleculares que se sintetizan de forma predominante en plantas, pero no en otros eucariotas, pertenecen a la familia sHSP (small heat-shock proteins). Se trata de una familia inusualmente compleja y heterogénea, cuyos miembros son de pequeño tamaño (16-42 kD) y poseen un dominio “alfa-cristalina” muy conservado. Estas proteínas están implicadas en protección frente a estrés abiótico mediante la estabilización de proteínas y membranas, si bien su mecanismo de acción se conoce de forma incompleta. A pesar del evidente potencial aplicado de las proteínas sHSP, son muy escasos los estudios realizados hasta el momento con un enfoque netamente biotecnológico. Por otra parte, casi todos ellos se han llevado a cabo en especies herbáceas de interés agronómico o en especies modelo, como Arabidopsis thaliana. De ahí que las sHSP de arbóreas hayan sido mucho menos caracterizadas estructural y funcionalmente, y ello a pesar del interés económico y ecológico de los árboles y de su prolongada exposición vital a múltiples factores estresantes. La presente Tesis Doctoral se centra en el estudio de sHSP de varias especies arbóreas de interés económico. El escrutinio exhaustivo de genotecas de cDNA de órganos vegetativos nos ha permitido identificar y caracterizar los componentes mayoritarios de tallo en dos especies productoras de madera noble: nogal y cerezo. También hemos caracterizado la familia completa en chopo, a partir de su secuencia genómica completa. Mediante expresión heteróloga en bacterias, hemos analizado el efecto protector de estas proteínas in vivo frente a distintos tipos de estrés abiótico, relevantes para el sector productivo. Los resultados demuestran que las proteínas sHSP-CI: (i) aumentan la viabilidad celular de E.coli frente a casi todos estos factores, aplicados de forma individual o combinada; (ii) ejercen un rol estabilizador de las membranas celulares frente a condiciones adversas; (iii) sirven para mejorar la producción de otras proteínas recombinantes de interés comercial. El efecto protector de las proteínas sHSP-CI también ha sido analizado in planta, mediante la expresión ectópica de CsHSP17.5-CI en chopos. En condiciones normales de crecimiento no se han observado diferencias fenotípicas entre las líneas transgénicas y los controles, lo que demuestra que se pueden sobre-expresar estas proteínas sin efectos pleiotrópicos deletéreos. En condiciones de estrés térmico, por el contrario, los chopos transgénicos mostraron menos daños y un mejor crecimiento neto. En línea con lo anterior, las actividades biológicas de varias enzimas resultaron más protegidas frente a la inactivación por calor, corroborando la actividad chaperona propuesta para la familia sHSP y su conexión con la tolerancia al estrés abiótico. En lo que respecta a la multiplicación y propagación de chopo in vitro, una forma de cultivo que comporta estrés para las plantas, todas las líneas transgénicas se comportaron mejor que los controles en términos de producción de biomasa (callos) y regeneración de brotes, incluso en ausencia de estrés térmico. También se comportaron mejor durante su cultivo ex vitro. Estos resultados tienen gran potencial aplicado, dada la recalcitrancia de muchas especies vegetales de interés económico a la micropropagación y a la manipulación in vitro en general. Los resultados derivados de esta Tesis, aparte de aportar datos nuevos sobre el efecto protector de las proteínas sHSP citosólicas mayoritarias (clase CI), demuestran por vez primera que la termotolerancia de los árboles puede ser manipulada racionalmente, incrementando los niveles de sHSP mediante técnicas de ingeniería genética. Su interés aplicado es evidente, especialmente en un escenario de calentamiento global. ABSTRACT Abiotic stress produces considerable economic losses in the forest sector, with extreme temperature and drought being amongst the most relevant factors. As sessile organisms, plants have acquired molecular strategies to detect and recognize stressful factors and activate appropriate responses. A wealth of evidence has correlated such responses with the massive induction of proteins belonging to the molecular chaperone family. Molecular chaperones are proteins which interact with incorrectly folded proteins to help them refold to their native state. In contrast to other eukaryotes, the most prominent stress-induced molecular chaperones of plants belong to the sHSP (small Heat Shock Protein) family. sHSPs are a widespread and diverse class of molecular chaperones that range in size from 16 to 42k Da, and whose members have a highly conserved “alpha-crystallin” domain. sHSP proteins play an important role in abiotic stress tolerance, membrane stabilization and developmental processes. Yet, their mechanism of action remains largely unknown. Despite the applied potential of these proteins, only a few studies have addressed so far the biotechnological implications of this protein family. Most studies have focused on herbaceous species of agronomic interest or on model species such as Arabidopsis thaliana. Hence, sHSP are poorly characterized in long-lived woody species, despite their economic and ecological relevance. This Thesis studies sHSPs from several woody species of economic interest. The most prominent components, namely cytosolic class I sHSPs, have been identified and characterized, either by cDNA library screening (walnut, cherry) or by searching the complete genomic sequence (poplar). Through heterologous bacterial expression, we analyzed the in vivo protective effects of selected components against abiotic stress. Our results demonstrate that sHSP-CI proteins: (i) protect E. coli cells against different stressful conditions, alone or combined; (ii) stabilize cell membranes; (iii) improve the production of other recombinant proteins with commercial interest. The effects of CsHSP17.5-CI overexpression have also been studied in hybrid poplar. Interestingly, the accumulation of this protein does not have any appreciable phenotypic effects under normal growth conditions. However, the transgenic poplar lines showed enhanced net growth and reduced injury under heat-stress conditions compared to vector controls. Biochemical analysis of leaf extracts revealed that important enzyme activities were more protected in such lines against heat-induced inactivation than in control lines, lending further support to the chaperone mode of action proposed for the sHSP family. All transgenic lines showed improved in vitro and ex vitro performance (calli biomass, bud induction, shoot regeneration) compared to controls, even in the absence of thermal stress. Besides providing new insights on the protective role of HSP-CI proteins, our results bolster the notion that heat stress tolerance can be readily manipulated in trees through genetic engineering. The applied value of these results is evident, especially under a global warming scenario.

Optimización de parámetros fermentativos de calidad en vinos tintos de zonas cálidas

El aumento progresivo de la temperatura media anual y el déficit hídrico están provocando importantes cambios en la composición y la maduración de la uva, que repercuten directamente sobre el proceso fermentativo y, por ende, sobre la calidad del vino elaborado. En este trabajo se evalúan diferentes estrategias para la reducción del grado alcohólico, la mejora del color del vino y su estabilidad, y el incremento y la persistencia aromática. Mediante el empleo de levaduras con ineficiencia glicolítica se lograron reducciones medias en el grado alcohólico de entre 0.3 y 1.7 % v/v, mientras que con las fermentaciones secuenciales la máxima reducción lograda fue de 3.3 y 3.4 % v/v al combinar las cepas 938 (Schizosaccharomyces pombe) y 7013 (Torulaspora delbrueckii) con la 7VA (Saccharomyces cerevisiae). Al aplicar un tratamiento térmico sobre el inóculo, la TP2A(16) mostró una reducción media significativa en el grado alcohólico de 1 % v/v. El principal inconveniente en todas las técnicas empleadas para reducir el grado alcohólico fue la falta de repetibilidad en los resultados obtenidos. Por otra parte, la aplicación de altas presiones sobre uva despalillada resultó efectiva como tratamiento de pasteurización y como potenciador de la extracción de polifenoles, logrando un incremento en el contenido medio de antocianos totales del 12.4-18.5 %. La adición de flavonoides al mosto estimuló la formación de pigmentos estables como resultado de su condensación con antocianos mediada por acetaldehído. Con el empleo de Torulaspora delbrueckii en fermentación secuencial fue posible incrementar la producción de diacetilo y acetato de 2-feniletilo, además de la síntesis de un nuevo compuesto, el 3-etoxi-1-propanol. Sin embargo, su aportación sobre el color fue nula, así que debería combinarse con una cepa de Saccharomyces cerevisiae con buena formación de pigmentos piranoantociánicos. El empleo de Schizosaccharomyces pombe (938, V1) y Torulaspora delbrueckii (1880) en fermentaciones secuenciales y mixtas con Saccharomyces cerevisiae permitió mejorar el perfil sensorial del vino tinto mediante la mayor síntesis de polioles y la potenciación de aromas frutales, florales y herbáceos, e incrementar la estabilidad de la materia colorante al favorecer la formación de vitisinas y piranoantocianos vinilfenólicos. La crianza sobre lías en barrica a partir de levaduras seleccionadas, puede mejorar la complejidad y persistencia aromática del vino tinto, aunque sin grandes cambios en el color. ABSTRACT The progressive increase in annual average temperature, along with water deficit, is causing significant changes in grape composition and in its maturation, which directly affects the fermentative process and hence alters wine quality. In this work, different strategies for reducing the alcoholic strength, improve wine color and its stability, and increase aromatic complexity and its persistence, are evaluated. By using yeasts with glycolytic inefficiency, it was possible to achieve mean reductions between 0.3 and 1.7 % v/v in the alcoholic strength, while sequential fermentations allowed a maximum reduction of 3.3 and 3.4 % v/v by combining strains 938 (Schizosaccharomyces pombe) and 7013 (Torulaspora delbrueckii) with 7VA (Saccharomyces cerevisiae). When applying a heat shock treatment on the inoculum, only TP2A(16) strain showed a significant mean reduction of 1 % v/v in the alcohol content, compared with the control. The main drawback in all the techniques used to reduce the alcohol content was the lack of repeatability in the results. Moreover, the application of high pressures on destemmed grapes was effective as pasteurization treatment and also as enhancer of polyphenol extraction, achieving an increase of 12.4-18.5% in the average content of total anthocyanins. As expected, addition of flavonoids to the must, stimulated the formation of stable pigments, mainly as a result of condensation reactions between anthocyanins and flavanols mediated by acetaldehyde. With the use of Torulaspora delbrueckii strains in sequential fermentation with Saccharomyces cerevisiae, it was possible to increase the production of diacetyl and 2-phenylethyl acetate, besides the synthesis of a new compound: 3-ethoxy-1-propanol. The use of Schizosaccharomyces pombe (938, V1) and Torulaspora delbrueckii (1880) strains in sequential and mixed fermentations with Saccharomyces cerevisiae improved the sensory profile of red wine by increasing polyols synthesis and enhancing fruity, floral and herbaceous aromas, and it also increased the stability of the coloring matter by favouring vitisins and vinylphenolic pyranoanthocyanins formation. Ageing on lees in barrels from selected yeasts can improve the complexity and aromatic persistence of red wine, without major changes in the color.

Exploring new roles for the rpoS gene in the survival and virulence of the fire blight pathogen Erwinia amylovora

Erwinia amylovora causes fire blight in economically important plants of the family Rosaceae. This bacterial pathogen spends part of its life cycle coping with starvation and other fluctuating environmental conditions. In many Gram-negative bacteria, starvation and other stress responses are regulated by the sigma factor RpoS. We obtained an E. amylovora rpoS mutant to explore the role of this gene in starvation responses and its potential implication in other processes not yet studied in this pathogen. Results showed that E. amylovora needs rpoS to develop normal starvation survival and viable but nonculturable (VBNC) responses. Furthermore, this gene contributed to stationary phase cross-protection against oxidative, osmotic, and acid stresses and was essential for cross-protection against heat shock, but nonessential against acid shock. RpoS also mediated regulation of motility, exopolysaccharide synthesis, and virulence in immature loquats, but not in pear plantlets, and contributed to E. amylovora survival in nonhost tissues during incompatible interactions. Our results reveal some unique roles for the rpoS gene in E. amylovora and provide new knowledge on the regulation of different processes related to its ecology, including survival in different environments and virulence in immature fruits.

Nonlocal electron heat relaxation in a plasma shock at arbitrary ionization number

A recently obtained nonlocal expression for the electron heat flux valid for arbitrary ionization numbers Z is used to study the structure of a plane shock wave in a fully ionized plasma. Nonlocal effects are only important in the foot of the electronic preheating region, where the electron temperature gradient is the steepest. The results are quantified as a function of a characteristic Knudsen number of that region. This work also generalizes to arbitrary values of Z previous results on plasma shock wave structure.

Compression of matter by hypersphsericla shock waves

A novel compression scheme is proposed, in which hollow targets with specifically curved structures initially filled with uniform matter, are driven by converging shock waves. The self-similar dynamics is analyzed for converging and diverging shock waves. The shock-compressed densities and pressures are much higher than those achieved using spherical shocks due to the geometric accumulation. Dynamic behavior is demonstrated using two-dimensional hydrodynamic simulations. The linear stability analysis for the spherical geometry reveals a new dispersion relation with cut-off mode numbers as a function of the specific heat ratio, above which eigenmode perturbations are smeared out in the converging phase.