Improved walnut mass micropropagation through the combined use of phloroglucinol and FeEDDHA

Despite the socioeconomic importance of walnut trees, poor rooting and recalcitrance to in vitro culture have hampered the establishment of high-yield clonal plantations. To improve walnut micropropagation, we introduced several modifications to current methods and evaluated the effects on microshoot performance and acclimatization. Nine selected genotypes (13-year-old trees) of the commercial hybrid Juglans major 209 x J. regia were cultured in vitro on DKW-C medium supplemented with 4.4 µM BA and 50 µM IBA. A protocol was developed that relies on the use of 0.40 mM phloroglucinol during shoot multiplication, 0.20 mM previous root induction, and 6.81 mg/L Fe3+ (FeEDDHA). Moreover, the addition of 83.2 µM glucose during the root expression phase significantly improved plant survival during acclimatization. Phloroglucinol promoted microshoot elongation but inhibited rooting, especially at concentrations above 0.40 mM. Replacing FeEDTA by FeEDDHA diminished chlorotic symptoms and improved rooting, with up to 90% microshoots developing viable roots. Likewise, glucose was more efficient than sucrose or fructose in promoting plant survival. At the proposed working concentrations, neither glucose nor FeEDDHA caused any noticeable deleterious effect on walnut micropropagation. Microscopic analysis revealed the physical continuity between adventitious roots and stem pericycles. Analysis of leaf genomic DNA with eight polymorphic microsatellite markers was supportive of the clonal fidelity and genetic stability of the micropropagated material. Successful clonal plantations (over 5,800 ramets) have been established by applying this protocol.

Seasonal evolution of soil respiration in a mixed forestry plantation of walnuts (Juglans × intermedia Carr.) and alders (Alnus cordata (Loisel.) Duby) in Domaine de Restinclières (France)

The ecological intensification of crops is proposed as a solution to the growing demand of agricultural and forest resources, in opposition to intensive monocultures. The introduction of mixed cultures as mixtures between nitrogen fixing species and non nitrogen fixing species intended to increase crop yield as a result of an improvement of the available nitrogen and phosphorus in soil. Relationship between crops have received little attention despite the wide range of advantages that confers species diversity to these systems, such as increased productivity, resilience to disruption and ecological sustainability. Forests and forestry plantations can develop an important role in storing carbon in their tissues, especially in wood which become into durable product. A simplifying parameter to analyze the amount allocated carbon by plantation is the TBCA (total belowground carbon allocation), whereby, for short periods and mature plantations, is admitted as the subtraction between soil carbon efflux and litterfall. Soil respiration depends on a wide range of factors, such as soil temperature and soil water content, soil fertility, presence and type of vegetation, among others. The studied orchard is a mixed forestry plantation of hybrid walnuts(Juglans × intermedia Carr.) for wood and alders (Alnus cordata (Loisel.) Duby.), a nitrogen fixing specie through the actinomycete Frankia alni ((Woronin, 1866) Von Tubeuf 1895). The study area is sited at Restinclières, a green area near Montpellier (South of France). In the present work, soil respiration varied greatly throughout the year, mainly influenced by soil temperature. Soil water content did not significantly influence the response of soil respiration as it was constant during the measurement period and under no water stress conditions. Distance between nearest walnut and measurement was also a highly influential factor in soil respiration. Generally there was a decreasing trend in soil respiration when the distance to the nearest tree increased. It was also analyzed the response of soil respiration according to alder presence and fertilizer management (50 kg N·ha-1·año-1 from 1999 to 2010). None of these treatments significantly influenced soil respiration, although previous studies noticed an inhibition in rates of soil respiration under fertilized conditions and high rates of available nitrogen. However, treatments without fertilization and without alder presence obtained higher respiration rates in those cases with significant differences. The lack of significant differences between treatments may be due to the high coefficient of variation experienced by soil respiration measurements. Finally an asynchronous fluctuation was observed between soil respiration and litterfall during senescence period. This is possibly due to the slowdown in the emission of exudates by roots during senescence period, which are largely related to microbial activity.

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.

Hyperspectral to multispectral imaging for detection of tree nuts and peanut traces in wheat flour

In current industrial environments there is an increasing need for practical and inexpensive quality control systems to detect the foreign food materials in powder food processing lines. This demand is especially important for the detection of product adulteration with traces of highly allergenic products, such as peanuts and tree nuts. Manufacturing industries dealing with the processing of multiple powder food products present a substantial risk for the contamination of powder foods with traces of tree nuts and other adulterants, which might result in unintentional ingestion of nuts by the sensitised population. Hence, the need for an in-line system to detect nut traces at the early stages of food manufacturing is of crucial importance. In this present work, a feasibility study of a spectral index for revealing adulteration of tree nut and peanut traces in wheat flour samples with hyperspectral images is reported. The main nuts responsible for allergenic reactions considered in this work were peanut, hazelnut and walnut. Enhanced contrast between nuts and wheat flour was obtained after the application of the index. Furthermore, the segmentation of these images by selecting different thresholds for different nut and flour mixtures allowed the identification of nut traces in the samples. Pixels identified as nuts were counted and compared with the actual percentage of peanut adulteration. As a result, the multispectral system was able to detect and provide good visualisation of tree nut and peanut trace levels down to 0.01% by weight. In this context, multispectral imaging could operate in conjuction with chemical procedures, such as Real Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay to save time, money and skilled labour on product quality control. This approach could enable not only a few selected samples to be assessed but also to extensively incorporate quality control surveyance on product processing lines.

Hyperspectral to multispectral imaging for detection of tree nuts and peanut traces in wheat flour.

In current industrial environments there is an increasing need for practical and inexpensive quality control systems to detect the foreign food materials in powder food processing lines. This demand is especially important for the detection of product adulteration with traces of highly allergenic products, such as peanuts and tree nuts. Manufacturing industries dealing with the processing of multiple powder food products present a substantial risk for the contamination of powder foods with traces of tree nuts and other adulterants, which might result in unintentional ingestion of nuts by the sensitised population. Hence, the need for an in-line system to detect nut traces at the early stages of food manufacturing is of crucial importance. In this present work, a feasibility study of a spectral index for revealing adulteration of tree nut and peanut traces in wheat flour samples with hyperspectral images is reported. The main nuts responsible for allergenic reactions considered in this work were peanut, hazelnut and walnut. Enhanced contrast between nuts and wheat flour was obtained after the application of the index. Furthermore, the segmentation of these images by selecting different thresholds for different nut and flour mixtures allowed the identification of nut traces in the samples. Pixels identified as nuts were counted and with the actual percentage of peanut adulteration. As a result, the multispectral system was able to detect and provide good visualisation of tree nut and peanut trace levels down to 0.01% by weight. In this context, multispectral imaging could operate in conjuction with chemical procedures, such as Real Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay to save time, money and skilled labour on product quality control. This approach could enable not only a few selected samples to be assessed but also to extensively incorporate quality control surveyance on product processing lines.

Nuevos mecanismos moleculares de tolerancia a sequía y otros tipos de estrés abiótico en especies arbóreas de interés económico

Las masas forestales tienen una importancia colosal para nuestra sociedad y el conjunto de la biosfera. Estudios recientes a escala mundial indican que la sequía es el factor abiótico que más afecta a su crecimiento y supervivencia, seguida por las temperaturas extremas y la salinidad. Aunque comprender los mecanismos con que las especies arbóreas toleran estas formas de estrés tiene un interés aplicado evidente, dichos mecanismos se han estudiado mucho más en especies herbáceas modelo o de interés agronómico. Existen sin embargo diferencias notables entre ellas, como se demuestra en esta tesis y en otros trabajos recientes. Nuestro estudio se centra concretamente en la respuesta molecular del chopo –el sistema modelo forestal más desarrollado– al estrés abiótico, con particular énfasis en la sequía. Utilizando una estrategia proteómica y tratamientos controlados, hemos identificado componentes mayoritarios de dicha respuesta. Su participación en la misma se ha validado mediante análisis transcripcionales detallados utilizando tecnología qRT-PCR (PCR cuantitativa en tiempo real). Hemos identificado proteínas cuyo nexo funcional con mecanismos de tolerancia ya era conocido, como chaperonas moleculares sHSP o enzimas que atenúan el estrés oxidativo, pero también proteínas cuya relación funcional con el estrés es menos clara o incluso novedosa, como polifenol oxidasas (PPO), deshidrogenasas/reductasas de cadena corta (SDR), o bicupinas (BIC), entre otras. El cuerpo central de la tesis consiste en la caracterización detallada de una PPO inusual, cuya inducción por estrés hídrico se describe por vez primera. Estas enzimas están ampliamente distribuidas en plantas, si bien su número es muy variable de unas especies a otras. Algunas, como nogal, tienen un único gen, mientras que Arabidopsis no tiene ninguno. En la última versión del genoma de chopo hemos identificado un total de 12 miembros bona fide, corrigiendo trabajos previos, y hemos caracterizado su expresión individual ante diferentes situaciones de estrés controlado y tratamientos hormonales. La isoforma antedicha es el único miembro de la familia que responde claramente a la deshidratación. También responde a salinidad y a la mayor parte de tratamientos hormonales ensayados, pero no a daño mecánico o tratamientos con metil jasmonato. Esto la diferencia de enzimas homólogas presentes en otras especies de plantas, que se han relacionado experimentalmente con estrés biótico. Los patrones de acumulación de transcritos en árboles adultos son compatibles con un papel protector frente a la sequía. La integración de nuestros estudios funcionales y filogenéticos sugiere que la familia ha sufrido un proceso reciente de diversificación y neofuncionalización, siendo la protección frente a deshidratación su papel primigenio. Aunque se conoce la actividad bioquímica in vitro de este tipo de enzimas, sus sustratos naturales son esencialmente una incógnita. Mediante expresión heteróloga en Escherichia coli BL21(DE3) hemos detectado que la enzima de chopo es capaz de oxidar L-DOPA a dopaquinona, siendo menos activa frente a otros sustratos. Por otra parte, hemos demostrado su localización cloroplástica mediante transformación transitoria de protoplastos con fusiones a la proteína fluorescente YFP. Mediante la obtención de plantas transgénicas de A. thaliana hemos demostrado que la enzima de chopo aumenta considerablemente la tolerancia in vivo frente a la deshidratación y al estrés salino. El análisis fenotípico detallado de las líneas transgénicas, combinando múltiples metodologías, nos ha permitido sustanciar que la tolerancia tiene una base compleja. Esta incluye una mayor protección del sistema fotosintético, una capacidad antioxidante muy incrementada y la acumulación de solutos osmoprotectores como la prolina. Los análisis metabolómicos nos han permitido asociar la expresión de la proteína a la síntesis de un flavano no descrito previamente en A. thaliana, vinculando la enzima de chopo con la síntesis de fenilpropanoides. También hemos observado alteraciones en los niveles hormonales que podrían subyacer a efectos pleiotrópicos con interés aplicado, como un aumento consistente del tamaño de la planta o el acortamiento del ciclo de crecimiento. Además de aportar datos novedosos sobre la funcionalidad in vivo de esta familia de oxidasas, los resultados de esta tesis demuestran que los árboles son sistemas de estudio interesantes para caracterizar nuevas estrategias de tolerancia al estrés abiótico con potencial aplicado. ABSTRACT Forests masses have an extraordinary importance for our society and the biosphere. Recent worldwide studies indicate that drought is the abiotic factor that affects more their growing and survival, followed by extreme temperatures and salinity. The understanding of how the arboreal species tolerate the stress has an evident practical interest, but their mechanisms have been studied much more in herbaceous species or with agronomic interest. However, considerable differences exist between them, as this thesis and recent studies show. Our study is focused on the molecular response of the poplar –the more developed forestry model system- to abiotic stress, specifically focused in the drought. Using a proteomic strategy and controlled treatments, we have identified main components in such response. Its participation has been validated through transcriptional analysis using qRT-PCR technology. We have identified proteins whose functional connection with tolerance mechanisms were already known, as molecular chaperones sHSP or enzymes that attenuate the oxidative stress, but also some proteins whose functional relationship with the stress is less clear or even novel, as polifenol oxidases (PPO), short chain deshidrogenases/reductases (SDR), or bicupines (BIC), among others. The central body of the thesis consists of the detailed characterization of an unsual PPO, whose induction due to drought stress is first described. These enzymes are thoroughly distributed in plants, but their number of members is very variable among species. Some of them, as the walnut tree, have a single gene, while Arabidopsis has none. We have identified a total of 12 members in the last version of the poplar genome, correcting previous works, and have characterized their individual expression against different situations of controlled stress and hormone treatments. The aforementioned isoform is the only member of the family that responds clearly to the drought. It also reacts to salinity and the majority of hormonal treatments tested, but it does not respond to mechanical damage or treatments with methyl jasmonate. This is the difference with homologue enzymes present in other plant species, which have been related experimentally with abiotic stress. The accumulation patterns of transcripts in adult trees are compatible with a protector role against drought. The integration of our functional and phylogenetic studies suggests that the family has suffered a recent process of diversification and neofunctionalization, being the protection against drought their original role. Although the in vitro biochemistry activity of this kind of enzymes is already known, their natural substracts are essentially a mystery. By means of heterologous expression of Escherichia coli BL21(DE3) we have detected that the enzyme of poplar is able to oxidize L-DOPA to dopaquinone, being less active against other substrates. Additionally, we have proven its chloroplastic location with transitory transformation of protoplasts with YFP protein fusion. By means of getting transgenic plants of A. thaliana, we have demonstrated that the poplar enzyme increases notably the in vivo tolerance against the drought and salinity stresses. The phenotypic analysis of the transgenic lines, and the use of multiple methodologies, allowed us to test the complexity of the tolerance. This includes a major protection of the photosynthetic system, a very increased antioxidant capacity and the accumulation of osmoprotectant solutes as the proline. The metabolic analysis has allowed to associate the protein expression with the synthesis of a Flavan non described previously in A. thalaiana, linking the enzyme of poplar with the synthesis of phenylpropanoids. We have observed alterations in the hormonal levels that could underlie pleiotropic effects with applied interest, as a consistent increase of the size of the plant and the reduction of the growth cycle. The results of this thesis, in addition to provide novel data about the in vivo functionality of the oxidase family, demonstrate that the trees are interesting systems of study to characterize new strategies of tolerance against abiotic stress with applied potential.

Is Microarray Analysis Really Useful and Sufficient to Diagnose Nut Allergy in the Mediterranean Area?

Background: Component-based diagnosis on multiplex platforms is widely used in food allergy but its clinical performance has not been evaluated in nut allergy. Objective: To assess the diagnostic performance of a commercial protein microarray in the determination of specific IgE (sIgE) in peanut, hazelnut, and walnut allergy. Methods: sIgE was measured in 36 peanut-allergic, 36 hazelnut-allergic, and 44 walnut-allergic patients by ISAC 112, and subsequently, sIgE against available components was determined by ImmunoCAP in patients with negative ISAC results. ImmunoCAP was also used to measure sIgE to Ara h 9, Cor a 8, and Jug r 3 in a subgroup of lipid transfer protein (LTP)-sensitized nut-allergic patients (positive skin prick test to LTP-enriched extract). sIgE levels by ImmunoCAP were compared with ISAC ranges. Results: Most peanut-, hazelnut-, and walnut-allergic patients were sensitized to the corresponding nut LTP (Ara h 9, 66.7%; Cor a 8, 80.5%; Jug r 3, 84% respectively). However, ISAC did not detect sIgE in 33.3% of peanut-allergic patients, 13.9% of hazelnut-allergic patients, or 13.6% of walnut-allergic patients. sIgE determination by ImmunoCAP detected sensitization to Ara h 9, Cor a 8, and Jug r 3 in, respectively, 61.5% of peanut-allergic patients, 60% of hazelnut-allergic patients, and 88.3% of walnut-allergic patients with negative ISAC results. In the subgroup of peach LTP?sensitized patients, Ara h 9 sIgE was detected in more cases by ImmunoCAP than by ISAC (94.4% vs 72.2%, P<.05). Similar rates of Cor a 8 and Jug r 3 sensitization were detected by both techniques. Conclusions: The diagnostic performance of ISAC was adequate for hazelnut and walnut allergy but not for peanut allergy. sIgE sensitivity against Ara h 9 in ISAC needs to be improved.