Environmentally benign Fe chelates in plant nutrition

The low solubility of iron (Fe) depresses plant growth in calcareous soils. In order to improve Fe availability, calcareous soils are treated with synthetic ligands, such as ethylenediaminetetraacetic acid (EDTA) and ethylenediimi-nobis(2-hydroxyphenyl)acetic acid (EDDHA). However, high expenses may hinder their use (EDDHA), and the recalcitrance of EDTA against biodegra-dation may increase the potential of cadmium (Cd) and lead (Pb) leaching. This study evaluated the ability of biodegradable ligands, i.e. different stereo-isomers of ethylenediaminedisuccinic acid (EDDS), to provide Fe for lettuce (Lactuca sativa L.) and ryegrass (Lolium perenne cv. Prego), their effects on uptake of other elements and solubility in soils and their subsequent effects on the activity of oxygen-scavenging enzymes in lettuce. Both EDTA and EDDHA were used as reference ligands. In unlimed and limed quartz sand both FeEDDS(S,S) and a mixture of stereo-isomers of FeEDDS (25% [S,S]-EDDS, 25% [R,R]-EDDS and 50% [S,R]/[R,S]-EDDS), FeEDDS(mix), were as efficient as FeEDTA and FeEDDHA in providing lettuce with Fe. However, in calcareous soils only FeEDDS(mix) was comparable to FeEDDHA when Fe was applied twice a week to mimic drip irrigation. The Fe deficiency increased the manganese (Mn) concentration in lettuce in both acidic and alkaline growth media, whereas Fe chelates depressed it. The same was observed with zinc (Zn) and copper (Cu) in acidic growth media. EDDHA probably affected the hormonal status of lettuce as well and thus depressed the uptake of Zn and Mn even more. The nutrient concentrations of ryegrass were only slightly affected by the Fe availability. After Fe chelate splitting in calcareous soils, EDDS and EDTA increased the solubility of Zn and Cu most, but only the Zn concentration was increased in lettuce. The availability of Fe increased the activity of oxygen-scavenging enzymes (ascorbate peroxidase, guaiacol peroxidase, catalase). The activity of Cu/ZnSOD (Cu/Zn superoxide dismutase) and MnSOD in lettuce leaves followed the concentrations of Zn and Mn. In acidic quartz sand low avail-ability of Fe increased the cobalt (Co) and nickel (Ni) concentrations in let-tuce, but Fe chelates decreased them. EDTA increased the solubility of Cd and Pb in calcareous soils, but not their uptake. The biodegradation of EDDS was not affected by the complexed element, and [S,S]-EDDS was biodegraded within 28 days in calcareous soils. EDDS(mix) was more recalcitrant, and after 56 days of incubation water-soluble elements (Fe, Mn, Zn, Cu, Co, Ni, Cd and Pb) corresponded to 10% of the added EDDS(mix) concentration.

Design, synthesis and characterization of sequence-specific DNA-cleaving metalloproteins

This thesis describes research pursued in two areas, both involving the design and synthesis of sequence specific DNA-cleaving proteins. The first involves the use of sequence-specific DNA-cleaving metalloproteins to probe the structure of a protein-DNA complex, and the second seeks to develop cleaving moieties capable of DNA cleavage through the generation of a non-diffusible oxidant under physiological conditions.

Chapter One provides a brief review of the literature concerning sequence-specific DNA-binding proteins. Chapter Two summarizes the results of affinity cleaving experiments using leucine zipper-basic region (bZip) DNA-binding proteins. Specifically, the NH_2-terminal locations of a dimer containing the DNA binding domain of the yeast transcriptional activator GCN4 were mapped on the binding sites 5'-CTGACTAAT-3' and 5'ATGACTCTT- 3' using affinity cleaving. Analysis of the DNA cleavage patterns from Fe•EDTA-GCN4(222-281) and (226-281) dimers reveals that the NH_2-termini are in the major groove nine to ten base pairs apart and symmetrically displaced four to five base pairs from the central C of the recognition site. These data are consistent with structural models put forward for this class of DNA binding proteins. The results of these experiments are evaluated in light of the recently published crystal structure for the GCN4-DNA complex. Preliminary investigations of affinity cleaving proteins based on the DNA-binding domains of the bZip proteins Jun and Fos are also described.

Chapter Three describes experiments demonstrating the simultaneous binding of GCN4(226-281) and 1-Methylimidazole-2-carboxamide-netropsin (2-ImN), a designed synthetic peptide which binds in the minor groove of DNA at 5'-TGACT-3' sites as an antiparallel, side-by-side dimer. Through the use of Fe•EDTA-GCN4(226-281) as a sequence-specific footprinting agent, it is shown that the dimeric protein GCN4(226-281) and the dimeric peptide 2- ImN can simultaneously occupy their common binding site in the major and minor grooves of DNA, respectively. The association constants for 2-ImN in the presence and in the absence of Fe•EDTA-GCN4(226-281) are found to be similar, suggesting that the binding of the two dimers is not cooperative.

Chapter Four describes the synthesis and characterization of PBA-β-OH-His- Hin(139-190), a hybrid protein containing the DNA-binding domain of Hin recombinase and the putative iron-binding and oxygen-activating domain of the antitumor antibiotic bleomycin. This 54-residue protein, comprising residues 139-190 of Hin recombinase with the dipeptide pyrimidoblamic acid-β-hydroxy-L-histidine (PBA-β-OH-His) at the NH2 terminus, was synthesized by solid phase methods. PBA-β-OH-His-Hin(139- 190) binds specifically to DNA at four distinct Hin binding sites with affinities comparable to those of the unmodified Hin(139-190). In the presence of dithiothreitol (DTT), Fe•PB-β-OH-His-Hin(139-190) cleaves DNA with specificity remarkably similar to that of Fe•EDTA-Hin(139-190), although with lower efficiency. Analysis of the cleavage pattern suggests that DNA cleavage is mediated through a diffusible species, in contrast with cleavage by bleomycin, which occurs through a non-diffusible oxidant.

The changes in various iron compounds in culture media for algae. [Translation from: Archiv fur Hydrobiologie Supplemente Band 38(1/2) 151-169, 1970.]

The purpose of this work is a contribution to the quantitative record of the use of iron by planktonic algae. Preliminary experiments with Chlorella to determine the rate of iron intake in the presence of inorganic sources of iron did not produce the desired result. The crucial point of this work is the investigation of the influence of various external factors on the stability of FeEDTA (FeEDTA = Ferric(III)-compound of ethylene-diamine tetra-acetic acid), since this compound appears to be particularly well-suited as a source of iron for planktonic algae (e.g. TAMIYA et al. 1953). Cultures of Chlorella fusca in a light thermostat were used in experimental research. Methods and results are discussed.

The Domon Family of Plant Plasma Membrane B-Type Cytochromes: Heterologous Expression, Biochemical Characterization and Physiological Roles in Vivo

The DOMON domain is a domain widespread in nature, predicted to fold in a β-sandwich structure. In plants, AIR12 is constituted by a single DOMON domain located in the apoplastic space and is GPI-modified for anchoring to the plasma membrane. Arabidopsis thaliana AIR12 has been heterologously expressed as a recombinant protein (recAtAIR12) in Pichia pastoris. Spectrophotometrical analysis of the purified protein showed that recAtAir12 is a cytochrome b. RecAtAIR12 is highly glycosylated, it is reduced by ascorbate, superoxide and naftoquinones, oxidised by monodehydroascorbate and oxygen and insensitive to hydrogen peroxide. The addition of recAtAIR12 to permeabilized plasma membranes containing NADH, FeEDTA and menadione, caused a statistically significant increase in hydroxyl radicals as detected by electron paramagnetic resonance. In these conditions, recAtAIR12 has thus a pro-oxidant role. Interestingly, AIR12 is related to the cytochrome domain of cellobiose dehydrogenase which is involved in lignin degradation, possibly via reactive oxygen species (ROS) production. In Arabidopsis the Air12 promoter is specifically activated at sites where cell separations occur and ROS, including •OH, are involved in cell wall modifications. air12 knock-out plants infected with Botrytis cinerea are more resistant than wild-type and air12 complemented plants. Also during B. cinerea infection, cell wall modifications and ROS are involved. Our results thus suggest that AIR12 could be involved in cell wall modifying reactions by interacting with ROS and ascorbate. CyDOMs are plasma membrane redox proteins of plants that are predicted to contain an apoplastic DOMON fused with a transmembrane cytochrome b561 domain. CyDOMs have never been purified nor characterised. The trans-membrane portion of a soybean CyDOM was expressed in E. coli but purification could not be achieved. The DOMON domain was expressed in P. pastoris and shown to be itself a cytochrome b that could be reduced by ascorbate.

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.

Biotecnología forestal aplicada a la producción de madera de nogal

Los nogales son especies pertenecientes a la familia Juglandaceae, ampliamente distribuidos por las zonas templadas y subtropicales del planeta. Son apreciados desde la antigüedad por la calidad de sus frutos y su madera. Su grado de domesticación es relativamente bajo comparado con cultivos alimenticios, e incluso respecto a otras especies forestales. Aunque el mercado de la madera de nogal suele mover grandes cantidades de dinero, su producción está basada principalmente en explotaciones extensivas, creciendo bajo sistemas poco tecnificados. Algunos intentos se han realizado en los EUA y Europa para la obtención de variedades madereras de nogal, pero ya sea por sus prolongados ciclos biológicos, por la complejidad genética de los caracteres sobre los que habría que incidir, por la escasa experiencia acumulada en programas de mejoramiento o por las limitaciones de los protocolos comerciales de reproducción asexual disponibles, lo cierto es que no existen genotipos seleccionados y destinados para tal fin. Poseer la capacidad de reproducir asexualmente los genotipos selectos es el complemento necesario de los programas de mejoramiento genético. Igualmente constituye la base de cualquier sistema productivo intensivo. Los sistemas de propagación vegetativa tradicionales en la familia Juglandaceae, además de ser inefectivos para la producción de elevados volúmenes de plantas, implican una gran complejidad en su ejecución y sus resultados suelen ser impredecibles. La micropropagación se plantea como la mejor alternativa para superar las dificultades de estas técnicas. Sin embargo, los nogales son considerados como altamente recalcitrantes al cultivo de tejidos, lo que provoca que sólo muy pocos genotipos sean propagados de forma comercial. Varias fases de la micropropagación de los nogales son especialmente conflictivas, e inciden de manera individual, y en su conjunto, sobre el resultado final. El control de los contaminantes microbianos, junto con la emisión de sustancias fenólicas y el decaimiento de los cultivos dificultan el establecimiento in vitro de la gran mayoría de los genotipos. La re-emergencia de microorganismos durante la proliferación es una fuente de pérdidas importante que también puede conducir al fracaso de la micropropagación. Las bajas tasas de enraizamiento y la elevada mortalidad registrada durante la aclimatación, unidas a factores genéticos, terminan por limitar la utilización comercial de esta tecnología a unos pocos genotipos. Con el objetivo de desarrollar un protocolo de micropropagación pre-comercial para el nogal híbrido maderero, se incidió en la solución de los principales problemas que dificultan la definición de ésta como una tecnología funcional para la producción de clones de ortetos selectos, al menos, para aquellos genotipos que pudieron ser establecidos in vitro. Así, aquí se presenta una metodología para el establecimiento in vitro que reduce la complejidad de esta fase y mejora el porcentaje de éxito durante la introducción. Igualmente se profundizó en el saneamiento de material contaminado y en el desarrollo de una herramienta que sirva para abordar el control de la re-emergencia microbiana durante la proliferación. También se analizan los elementos claves de este protocolo que garantizan la obtención de microbrotes de calidad enraizables, potencialmente capaces de soportar el paso a condiciones ex vitro. La evaluación de aspectos como la sustitución del FeEDTA por el FeEDDHA como fuente hierro, la introducción del Floroglucinol en el medio de cultivo de proliferación y la determinación de una fuente de carbono adecuada para la formación de las raíces, junto con un manejo adecuado de la fase previa a la pre-inducción radical, fueron determinantes en la micropropagación de hasta 14 genotipos de nogal híbrido maderero. Adicionalmente, la realización de un análisis detallado del sistema radical permitió comprobar que la metodología propuesta favorece la producción de raíces que están conectadas vascularmente con el tallo, lo que unido a la presencia de estomas morfológicamente normales, permite reducir las pérdidas por estas causas y favorece el proceso de endurecimiento. Como complemento del programa de mejora y de clonación, se abordó el desarrollo de una herramienta que permitiera identificar y diferenciar las selecciones. Buscando combinar la simplicidad de la técnica con el máximo poder discriminativo posible, se eligió el uso de marcadores genéticos del tipo SSR. En una primera aproximación, se evaluó la conveniencia de emplear primers previamente publicados (Woeste et al. 2002) y utilizados en varias especies de la familia Juglandaceae (Dangl et al. 2005, Victory et al. 2006, Ross-Davis y Woeste 2008, entre otros autores). A pesar de que la mayoría de las parejas de cebadores evaluadas rindieron productos de amplificación interpretables, su capacidad de clasificación conjunta fue muy reducida y de uso muy limitado en una población afectada por un alto grado de parentesco. Por esta razón fue necesario desarrollar una batería de marcadores microsatélites diseñados específicamente para el nogal híbrido maderero. De las 700 regiones secuenciadas, finalmente 24 parejas demostraron ser funcionales y lo suficientemente polimórficas como para discriminar entre medios hermanos. Al ser utilizadas 10 de las nuevas parejas de primers, junto con 2 de la genoteca desarrollada por Woeste et al. (2002), en una población diferente fue posible genotipar con una PID del orden de 10-11, lo que abre la posibilidad de utilizar este set de marcadores de novo dentro de la familia Juglandaceae.

Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease

The core enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several σ subunits. To characterize the proximity between σ70, the major σ for transcription of the growth-related genes, and the core enzyme subunits (α2ββ′), we analyzed the protein-cutting patterns produced by a set of covalently tethered FeEDTA probes [FeBABE: Fe (S)-1-(p-bromoacetamidobenzyl)EDTA]. The probes were positioned in or near conserved regions of σ70 by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581. Each FeBABE-conjugated σ70 was bound to the core enzyme, which led to cleavage of nearby sites on the β and β′ subunits (but not α). Unlike the results of random cleavage [Greiner, D. P., Hughes, K. A., Gunasekera, A. H. & Meares, C. F. (1996) Proc. Natl. Acad. Sci. USA 93, 71–75], the cut sites from different probe-modified σ70 proteins are clustered in distinct regions of the subunits. On the β subunit, cleavage is observed in two regions, one between residues 383 and 554, including the conserved C and Rif regions; and the other between 854 and 1022, including conserved region G, regions of ppGpp sensitivity, and one of the segments forming the catalytic center of RNA polymerase. On the β′ subunit, the cleavage was identified within the sequence 228–461, including β′ conserved regions C and D (which comprise part of the catalytic center).

Effects of iron additions on filament growth and productivity of the cyanobacterium Lyngbya majuscula

The bioavailability of iron, in combination with essential macronutrients such as phosphorus, has been hypothesised to be linked to nuisance blooms of the toxic cyanobacterium Lyngbya majuscula. The present laboratory study used two biological assay techniques to test whether various concentrations of added iron (inorganic and organically chelated) enhanced L. majuscula filament growth and productivity (C-14-bicarbonate uptake rate). Organically chelated iron (FeEDTA) with adequate background concentrations of phosphorus and molybdenum caused the largest increases (up to 4.5 times the control) in L. majuscula productivity and filament growth. The addition of inorganic iron (without added phosphorus or molybdenum) also stimulated L. majuscula filament growth. However, overall the FeEDTA was substantially and significantly more effective in promoting L. majuscula growth than inorganic iron (FeCl3). The organic chelator (EDTA) alone and molybdenum alone also enhanced L. majuscula growth but to a lesser extent than the chelated iron. The results of the present laboratory study support the hypothesis that iron and chelating organic compounds may be important in promoting blooms of L. majuscula in coastal waters of Queensland, Australia.