Phytoremediation of mercury by terrestrial plants

Mercury (Hg) pollution is a global environmental problem. Numerous Hg-contaminated sites exist in the world and new techniques for remediation are urgently needed. Phytoremediation, use of plants to remove pollutants from the environment or to render them harmless, is considered as an environment-friendly method to remediate contaminated soil in-situ and has been applied for some other heavy metals. Whether this approach is suitable for remediation of Hg-contaminated soil is, however, an open question. The aim of this thesis was to study the fate of Hg in terrestrial plants (particularly the high biomass producing willow, Salix spp.) and thus to clarify the potential use of plants to remediate Hg-contaminated soils. Plants used for phytoremediation of Hg must tolerate Hg. A large variation (up to 30-fold difference) was detected among the six investigated clones of willow in their sensitivity to Hg as reflected in their empirical toxicity threshold (TT95b), the maximum unit toxicity (UTmax) and EC50 levels. This gives us a possibility to select Hg-tolerant willow clones to successfully grow in Hgcontaminated soils for phytoremediation. Release of Hg into air by plants is a concern when using phytoremediation in practice. No evidence was found in this study that Hg was released to the air via shoots of willow, garden pea (Pisum sativum L. cv Faenomen), spring wheat (Triticum aestivum L. cv Dragon), sugar beet (Beta vulgaris L. cv Monohill), oil-seed rape (Brassica napus L. cv Paroll) and white clover (Trifolium repens L.). Thus, we conclude that the Hg burden to the atmosphere via phytoremediation is not increased. Phytoremediation processes are based on the ability of plant roots to accumulate Hg and to translocate it to the shoots. Willow roots were shown to be able to efficiently accumulate Hg in hydroponics, however, no variation in the ability to accumulate was found among the eight willow clones using CVAAS to analyze Hg content in plants. The majority of the Hg accumulated remained in the roots and only 0.5-0.6% of the Hg accumulation was translocated to the shoots. Similar results were found for the five common cultivated plant species mentioned above. Moreover, the accumulation of Hg in willow was higher when being cultivated in methyl-Hg solution than in inorganic Hg solution, whereas the translocation of Hg to the shoots did not differ. The low bioavailability of Hg in contaminated soil is a restricting factor for the phytoextraction of Hg. A selected tolerant willow clone was used to study whether iodide addition could increase the plant-accumulation of Hg from contaminated soil. Both pot tests and field trials were carried out. Potassium iodide (KI) addition was found to mobilize Hg in contaminated soil and thus increase the bioavailability of Hg in soils. Addition of KI (0.2–1 mM) increased the Hg concentrations up to about 5, 3 and 8 times in the leaves, branches and roots, respectively. However, too high concentrations of KI were toxic to plants. As the majority of the Hg accumulated in the roots, it might be unrealistic to use willow for phytoextraction of Hg in practice, even though iodide could enhance the phytoextraction efficiency. In order to study the effect of willow on various soil fractions of Hg-contaminated soil, a 5-step sequential soil extraction method was used. Both the largest Hg-contaminated fractions, i.e. the Hg bound to residual organic matter (53%) and sulphides (43%), and the residual fraction (2.5%), were found to remain stable during cultivations of willow. The exchangeable Hg (0.1%) and the Hg bound to humic and fulvic acids (1.1%) decreased in the rhizospheric soil, whereas the plant accumulation of Hg increased with the cultivation time. The sum of the decrease of the two Hg fractions in soils was approximately equal to the amount of the Hg accumulated in plants. Consequently, plants may be suitable for phytostabilization of aged Hg-contaminated soil, in which root systems trap the bioavailable Hg and reduce the leakage of Hg from contaminated soils.

Sintesi e proprietà di biochar da biomasse residuali

Il termine biochar definisce il prodotto solido derivante dalla pirolisi di un qualsiasi materiale organico, con lo specifico scopo di essere applicato nei suoli sia per fini agronomici che di gestione ambientale. Un suo utilizzo in maniera "responsabile" richiede però una piena comprensione delle sue proprietà e dei meccanismi che controllano la sua attività nel terreno, che dipendono dalla biomassa di partenza e dalle condizioni di sintesi tramite pirolisi. Infatti le condizioni di pirolisi, in particolare la temperatura di processo e il tempo di residenza, determinano biochar con caratteristiche differenti. In questo lavoro di tesi sono stati prodotti biochar da due diverse tipologie di biomassa residuale ampiamente disponibili (stocchi di mais e pollina). Per ciascuna biomassa sono state scelte tre condizioni di pirolisi (400°C x 20 minuti, 500°C x 10 minuti e 600°C x 5 minuti). Sui biochar ottenuti sono state effettuate le seguenti determinazioni: analisi elementare, Pirolisi‐GC‐MS, idrocarburi policiclici aromatici (IPA), acidi grassi volatili (VFA), azoto ammoniacale (N‐NH4 +), pH, conduttività elettrica e ritenzione idrica. Infine i biochar sintetizzati sono stati utilizzati per fare due test di germinazione per valutare l'effetto sulla formazione delle prime strutture di crescita delle plantule, tramite test di tossicità brevi con piastre Petri. Il primo test è stato condotto a concentrazione crescente di miscele acqua/biochar (2, 5, 40 e 100 g/L sulla base delle quantità di biochar utilizzate come ammendante nel suolo), sulla germinazione seguendo la metodologia normata dalla ISO 11269:2012. I semi utilizzati nel primo test sono stati quelli del crescione (Lepidium sativum L.) come specie dicotiledone, e del sorgo (Sorghum saccharatum M.) come monocotiledone. Il secondo saggio di tossicità eseguito è stato quello descritto dalla normativa in materia UNI 11357, valutando l'eventuale effetto di tossicità alla massima concentrazione delle varie tipologie di biochar, utilizzando come specie dicotiledoni il cetriolo (Cucumis sativus L.) ed il crescione (Lepidium sativum L.), come monocotiledone il sorgo (Sorghum saccharatum M.). Per i biochar da stocchi di mais, rappresentativi di biomasse erbacee e con diverso grado di carbonizzazione, non si osservano effetti apprezzabili alle condizioni di uso agricolo. Nel caso dei biochar da pollina si osservano invece inibizioni alla germinazione sin dalle concentrazioni più basse. In particolare, quello pirolizzato a 400°C mostra un potenziale effetto tossico più marcato, probabilmente associato ad un contenuto di IPA e VFA superiore a quello degli altri biochar.

Development of a biocrystallisation assay for examining effects of homeopathic preparations ursing cress seedlings

A major challenge in basic research into homeopathic potentisation is to develop bioassays that yield consistent results. We evaluated the potential of a seedling-biocrystallisation method. Cress seeds (Lepidium sativum L.) germinated and grew for 4 days in vitro in Stannum metallicum 30x or water 30x in blinded and randomized assignment. 15 experiments were performed at two laboratories. CuCl2-biocrystallisation of seedlings extracted in the homeopathic preparations was performed on circular glass plates. Resulting biocrystallograms were analysed by computerized textural image analysis. All texture analysis variables analysed yielded significant results for the homeopathic treatment; thus the texture of the biocrystallograms of homeopathically treated cress exhibited specific characteristics. Two texture analysis variables yielded differences between the internal replicates, most probably due to a processing order effect. There were only minor differences between the results of the two laboratories. The biocrystallisation method seems to be a promising complementary outcome measure for plant bioassays investigating effects of homeopathic preparations.

Dithiothreitol triggers photooxidative stress and fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in intact pea chloroplasts

In intact chloroplasts isolated from mature pea leaves (Pisum sativum L.), the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was rapidly fragmented into several products upon illumination in the presence of 1 mM dithiothreitol (DTT). Very similar effects on LSU stability could be observed when illuminated chloroplasts were poisoned with cyanide which, like DTT, inhibits important plastid antioxidant enzymes, or when a light-dependent hydroxyl radical-producing system was added to the incubation medium. Moreover, DTT-stimulated light degradation of LSU was markedly delayed in the presence of scavengers of active oxygen species (AOS). It is therefore suggested that light degradation of LSU in the presence of DTT is mainly due to inhibition of the chloroplast antioxidant defense system and the subsequent accumulation of AOS in intact organelles. When chloroplasts were isolated from nonsenescent or senescent leaves, LSU remained very stable upon incubation without DTT, indicating that the antioxidant system was still functional in the isolated chloroplasts during leaf ageing. Our data support the notion that AOS might be important for the degradation of Rubisco in vivo under oxidative stress.

Evaluación del rendimiento y calidad de cultivares de ajo colorado fertirrigados con nitrógeno

El objetivo de este trabajo fue diferenciar cultivares monoclonales de ajos colorados argentinos por sus características productivas, de calidad y respuesta a la fertilización nitrogenada en la región de Cuyo. Durante la campaña 2003 se realizó un ensayo en La Consulta, San Carlos, Mendoza, Argentina (950 msnm; 33° 42' S y 69° 04' W). El diseño experimental fue en franjas con parcelas divididas dispuestas en un diseño de bloques completos al azar de tres repeticiones en el cual las dosis de nitrógeno (75, 150, 225 y 300 kg de N·ha-1) incorporadas como SolUAN (30 % N) se asignaron a las parcelas principales y las subparcelas correspondieron a cinco clones de ajo: Fuego INTA, Sureño INTA, Gostoso INTA, Inco 30 y Rubí INTA. Se adicionó además un tratamiento control sin nitrógeno. En el análisis estadístico de los rendimientos (r2 = 0,81; cv%: 8,48 y p < 0,0001) se detectaron efectos significativos de las variables cultivares y dosis de fertilización nitrogenada pero no de la interacción de ambas. Se determinó que en suelos con contenidos medios de nitrógeno total (800 mg·kg-1), la dosis que maximiza los rendimientos es de 150 kg N·ha-1 en todas las cultivares salvo Gostoso cuyo valor crítico resultó mayor que el resto. El incremento de rendimiento atribuible a la fertirrigación con 150 kg N·ha-1 respecto del testigo fue: 45 % en Rubí (17,7 t· ha-1), 31 % en Fuego (15,3 t·ha-1), 19 % en Inco (15,4 t·ha-1) y 23 % en Sureño (13 t·ha-1). Gostoso aumentó un 21 % (13,8 t·ha-1) su rendimiento respecto del testigo con la dosis de 300 kg N·ha-1. El análisis de clasificación jerárquica permitió agrupar los genotipos según su potencial productivo, de mayor a menor, en tres clases: I: cv. Rubí ; II: cv. Inco 30 y cv. Fuego; III: cv. Sureño y Gostoso. La fertilización nitrogenada incrementó la manifestación de malformaciones en todas las cultivares respecto del testigo sin fertilización, excepto Sureño que presentó mayores anormalidades en el tratamiento testigo sin fertilizar.

Estudio de procesos de deshidratación industrial de ajo con la finalidad de preservar alicina como principio bioactivo

El ajo es ampliamente conocido por sus propiedades benéficas para la salud y por sus características sensoriales. Además de su consumo en fresco, se elaboran diversos subproductos industriales, entre ellos se destaca el ajo deshidratado. Este producto se emplea tanto para consumo como condimento como para la elaboración de fitofármacos. Un atributo de calidad buscado en el polvo de ajo es que tenga elevados contenidos de principios bioactivos, especialmente, para la elaboración de preparaciones galénicas. Los fitoquímicos presentes en el ajo son compuestos organoazufrados y el más importante y representativo del grupo es la alicina. Diversos autores coinciden en que las tabletas comerciales de ajo en polvo evidencian grandes fluctuaciones en el contenido de alicina. Estas pueden deberse a las diferencias en la materia prima y a las pérdidas durante el proceso de deshidratación industrial. El objetivo del trabajo fue evaluar los niveles de alicina en polvos de ajo obtenidos mediante distintos procesos de deshidratación. Se trabajó con ajo cv Sureño INTA. Los bulbos fueron seleccionados y los dientes pelados en forma manual. La pasta de ajo fue obtenida mediante molino helicoidal y homogeneizador. Los tratamientos fueron: deshidratación en Spray, en horno Terminador y en horno de deshidratación de Usos Múltiples. También se evaluó la eficacia del empleo de aditivos microencapsulantes (goma arábiga) en los distintos tratamientos de deshidratados: tipo Spray, Lecho Espumado. La evaluación de los procesos fue realizada mediante la medición de humedad y alicina (HPLC). Además, se cuantificó alicina en productos de ajo deshidratado disponibles en el mercado. De todos los tratamientos, el proceso de deshidratación Spray evidenció las menores pérdidas de alicina, en las condiciones bajo estudio. De lo que se desprende, que las condiciones para la deshidratación de alta temperatura, corto tiempo y empleo de aditivo microencapsulante resultaron propicios para preservar alicina.

Selenized garlic: a future prospect or already a current functional food?

In the last years, functional foods have awakened consumer, scientific and business interest. A commonly found vegetable in such kind of foods includes garlic (Allium sativum). By its ability for selenium (Se) bio-accumulation, garlic can turn into an attractive option of selenized food. Selenium is an essential micronutrient for many organisms including plants, animals, and humans. It is an important trace element due to its antioxidant properties and plays a main role in prevention of cancer and cardiovascular diseases. Nowadays, there is an increasing interest in the study of Se speciation due to the different roles that each species manifests in toxicological and nutrition fields. However, Se exhibits a narrow interval between toxicity and essentiality, which is puzzling toxicologists and alarming nutritionists and legislators. In the present review, an overview on the development of selenized garlic studies and its potential implementation in Argentine production is exposed. The development of novel foods with added value such us selenized garlic could be an attractive alternative for local market. Moreover, it becomes a good offering for factory owners, considering that Mendoza represents about 85% of total garlic production in the country.

Aceites saborizados con ajo (aderezos)

El ajo es una especie cultivada por sus propiedades terapéuticas, su significancia religiosa, su sabor y aroma. Por su alto aporte de flavonoides, compuestos polifenólicos y organoazufrados (OSCs) se encuentra entre los alimentos considerados saludables. Entre los efectos biológicos que posee se incluyen propiedades antimicrobianas, antidiabéticas, antimutagénicas y anticancerígenas. En la actualidad, existen en el mercado subproductos denominados aceites saborizados con ajo que de acuerdo a la legislación nacional vigente se comercializan bajo la denominación de aderezos sin ninguna diferenciación en cuanto al proceso de saborización empleado. Establecer el proceso de saborización utilizado resulta de importancia ya que de ello dependen tanto el perfil de compuestos bioactivos como las potenciales propiedades biológicas que estos ejerzan. Por otro lado, los aceites saborizados con ajo pueden ser empleados como medio para cocción mediante fritura. Numerosas variables se encuentran implicadas en este proceso culinario, existiendo condiciones de fritura óptimas para cada tipo de producto. De lo anteriormente expuesto surge el interés en caracterizar aceites saborizados con ajo en función de indicadores de calidad bromatológica, color y perfil cuali-cuantitativo de OSCs y en determinar posibles modificaciones durante su empleo como medio para la cocción mediante fritura. Para ello se adquirieron en el comercio local aceites saborizados con ajo en aceites vegetales de girasol, canola y oliva. Paralelamente, se obtuvieron aceites saborizados con ajo a escala de laboratorio utilizando dos métodos diferentes de saborización. Estos consistieron, por un lado en la adición de aceite destilado de ajo y por otro en la maceración de dientes de ajo fresco picado en un aceite vegetal. Posteriormente tanto los aceites comercializados como los obtenidos a escala de laboratorio, se sometieron a diferentes temperaturas de fritura (180 °C, 220 °C y 300 °C) durante 3 minutos. La calidad bromatológica se evaluó a través de las determinaciones de índice de peróxidos (IP) e índice de acidez (IA), el color a través del Sistema CIELAB y el perfil de OSCs mediante cromatografía gaseosa y cromatografía líquida. A partir de los indicadores de calidad bromatológica evaluados fue posible determinar el estado oxidativo de los aceites saborizados con ajo evaluados. El color estuvo determinado por las características y la presencia de pigmentos propios de cada aceite vegetal. Se evidenciaron diferencias tanto cuali- como cuantitativas en el perfil de OSCs, estableciendo que la metodología de HPLC es la más idónea para la detección de dichos compuestos. Se diferenciaron a los aceites saborizados con ajo bajo estudio en „aceites de ajo macerado‟ cuando contenían alicina, ajoeno y vinilditiinas y en „aceites destilados de ajo‟ por la presencia de sulfuros y polisulfuros. Al ser sometidos a condiciones de cocción mediante fritura, se observaron modificaciones en los indicadores de calidad bromatológica, color y perfil de OSCs. Estas modificaciones implicaron cambios en el IP, IA y color. En todos los casos las modificaciones observadas fueron dependientes del aceite vegetal evaluado. Respecto del perfil y concentración de OSCs, también se denotaron modificaciones, evidenciándose cierta inestabilidad de algunos compuestos. Otro aspecto importante a destacar es que aún a la temperatura más alta de fritura ensayada, los aceites saborizados con ajo aún contuvieron compuestos organoazufrados que evidencian importantes propiedades benéficas para la salud.

Effect of colchicine and amiprophos-methyl on the production of in vitro doubled haploid onion plants and correlation assessment between ploidy level and stomatal size

Doubled haploid onion (Allium cepa L.) plants allow the production of completely homozygous lines for a later production of hybrids. The haploid plants are normally produced using in vitro gynogenesis. The obtained haploid plantlets must be treated with different agents for doubling chromosomes. It is necessary to adjust the concentration and the length of treatment of the doubling agent. In this case, the effect of 250 and 500 mg.L-1 colchicine and 15.2; 30 and 60 mg.L- 1 amiprophos-methyl during 24 and 48 h was assessed over the rate of onion haploid plantlets chromosome doubling. The best duplication treatment was 250 mg.L-1 colchicine for 48 h, which yielded 100% of doubled haploid plants. On the other hand, a positive correlation resulted from the ploidy level and stomatal size, and a negative correlation between the level of ploidy and stomatal density. Significant differences between the stomatal length, width and density in haploid and doubled haploid plantlets were observed. An economical and quick method to test ploidy level in onion plantlets is proposed through the measurement of stomatal size and density.

Populations genomics analysis of legume host preference for specific rhizobial genotypes in the Rhizobium leguminosarum bv. viciae symbioses

Rhizobium leguminosarum bv. viciae establishes root nodule symbioses with several legume genera. Although most isolates are equally effective in establishing symbioses with all host genera, previous evidence suggests that hosts select specific rhizobial genotypes among those present in the soil. We have used population genomics to further investigate this observation. P. sativum, L. culinaris, V. sativa, and V. faba plants were used to trap rhizobia from a well-characterized soil, and pooled genomic DNAs from one-hundred isolates from each plant were sequenced. Sequence reads were aligned to the R. leguminosarum bv. viciae 3841 reference genome. High overall conservation of sequences was observed in all subpopulations, although several multigenic regions were absent from the soil population. A large fraction (16-22%) of sequence reads could not be recruited to the reference genome, suggesting that they represent sequences specific to that particular soil population. Although highly conserved, the 16S-23S rRNA gene region presented single nucleotide polymorphisms (SNPs) regarding the reference genome, but no striking differences could be found among plant-selected subpopulations. Plant-specific SNP patterns were, however, clearly observed within the nod gene cluster, supporting the existence of a plant preference for specific rhizobial genotypes. This was also shown after genome-wide analysis of SNP patterns.

Population genomics of host specificity in Rhizobium leguminosarum bv. viciae

Legumes establish a root-nodule symbiosis with soil bacteria collectively known as rhizobia. This symbiosis allows legumes to benefit from the nitrogen fixation capabilities of rhizobia and thus to grow in the absence of any fixed nitrogen source. This is especially relevant for Agriculture, where intensive plant growth depletes soils of useable, fixed nitrogen sources. One of the main features of the root nodule symbiosis is its specificity. Different rhizobia are able to nodulate different legumes. Rhizobium leguminosarum bv. viciae is able to establish an effective symbiosis with four different plant genera (Pisum, Lens, Vicia, Lathyrus), and any given isolate will nodulate any of the four plant genera. A population genomics study with rhizobia isolated from P. sativum, L. culinaris, V. sativa or V. faba, all originating in the same soil, showed that plants select specific genotypes from those available in that soil. This was demonstrated at the genome-wide level, but also for specific genes. Accelerated mesocosm studies with successive plant cultures provided additional evidence on this plant selection and on the nature of the genotypes selected. Finally, representatives from the major rhizobial genotypes isolated from these plants allowed characterization of the size and nature of the respective pangenome and specific genome compartments. These were compared to the different genotypes ?symbiotic and non-symbiotic?present in rhizobial populations isolated directly from the soil without plant intervention.

Effect of the Legume Plant Host on Rhizobial Soil Population Dynamics

Rhizobium leguminosarum bv viciae (Rlv) is a soil bacterium able to establish specific root-nodule symbioses with legumes of four different genera: Pisum, Vicia, Lens and Lathyrus. Rlv isolates from nodules of any of these legumes can nodulate any of them; however, it has been shown that plants select specific rhizobial genotypes from those present in the soil (1,2). We have previously shown this at the genomic level by following a population genomics approach. Pool genomic sequences from 100 isolates from each of four plant species: P. sativum, L. culinaris, V. faba and V. sativa, show different, specific profiles at the single nucleotide polymorphism (SNP) level for relevant genes. In this work, the extent of Rlv selection from a well-characterized soil population by different legume plant hosts: P. sativum, L. culinaris, V. faba and V. sativa, after a medium-term mesocosm study is described. Direct soil isolates from each of these mesocosm studies have been tested for specific rhizobial genes (glnII and fnrN) and symbiotic genes (nodC and nifH). Different populations were characterized further by Sanger sequencing of both the rpoB phylogenetic marker gene and the symbiotic genes nodC and nifH. The distribution and size of the rhizobial population for each legume host showed changes during the medium-term mesocosm study. Particularly, a non-symbiotic group of rhizobia was enriched by all four hosts, in contrast to the symbiotic rhizobia profile, which was specific for each legume plant host.

Mendel’s dwarfing gene: cDNAs from the Le alleles and function of the expressed proteins

The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA1, which is formed from GA20 by 3β-hydroxylation. This step, which limits GA1 biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3β-hydroxylase from lines of pea carrying the Le, le, le-3, and led alleles. The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le. The cDNA sequence from led, a mutant derived from an le line, contains both the le “mutation” and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in Escherichia coli. The expression product for the clone from Le converted GA9 to GA4, and GA20 to GA1, with Km values of 1.5 μM and 13 μM, respectively. The amino acid substitution in the clone from le increased Km for GA9 100-fold and reduced conversion of GA20 to almost nil. Expression products from le and le-3 possessed similar levels of 3β-hydroxylase activity, and the expression product from led was inactive. Our results suggest that the 3β-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.

A Calcium-Selective Channel from Root-Tip Endomembranes of Garden Cress1

A Ca2+ channel from root-tip endomembranes of garden cress (Lepidium sativum L.) (LCC1) was characterized using the planar lipid-bilayer technique. Investigation of single-channel recordings revealed that LCC1 is voltage gated and strongly rectifying. In symmetrical 50 mm CaCl2 solutions, the single-channel conductance was 24 picosiemens. LCC1 showed a moderate selectivity for Ca2+ over K+ (9.4:1) and was permeable for a range of divalent cations (Ca2+, Ba2+, and Sr2+). In contrast to Bryonia dioica Ca2+ channel 1, a Ca2+-selective channel from the endoplasmic reticulum of touch-sensitive tendrils, LCC1 showed no bursting channel activity and had a low open probability and mean open time (2.83 ms at 50 mV). Inhibitor studies demonstrated that LCC1 is blocked by micromolar concentrations of erythrosin B (inhibitor concentration for 50% inhibition [IC50] = 1.8 μm) and the trivalent cations La3+ (IC50 = 5 μm) and Gd3+ (IC50 = 10 μm), whereas verapamil showed no blocking effect. LCC1 may play an important role in the regulation of the cytoplasmic free Ca2+ concentration in root-tip and/or root-cap cells. The question of whether this ion channel is part of the gravitropic signal transduction pathway deserves further investigation.

Study of the 3-Hydroxy Eicosanoyl-Coenzyme A Dehydratase and (E)-2,3 Enoyl-Coenzyme A Reductase Involved in Acyl-Coenzyme A Elongation in Etiolated Leek Seedlings1

(R,S)-[1-14C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-14C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-14C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-14C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-14C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-14C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-14C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg−1 h−1, which is the highest ever reported for a plant system.