Aaron C. Asensio

Changes in the C/N balance caused by increasing external ammonium concentrations are driven by carbon and energy availabilities during ammonium nutrition in pea plants: the key roles of asparagine synthetase and anaplerotic enzymes

An understanding of the mechanisms underlying ammonium (NH(4)(+)) toxicity in plants requires prior knowledge of the metabolic uses for nitrogen (N) and carbon (C). We have recently shown that pea plants grown at high NH(4)(+) concentrations suffer an energy deficiency associated with a disruption of ionic homeostasis. Furthermore, these plants are unable to adequately regulate internal NH4(+) levels and the cell-charge balance associated with cation uptake. Herein we show a role for an extra-C application in the regulation of C-N metabolism in NH(4)(+) -fed plants. Thus, pea plants (Pisum sativum) were grown at a range of NH(4)(+) concentrations as sole N source, and two light intensities were applied to vary the C supply to the plants. Control plants grown at high NH(4)(+) concentration triggered a toxicity response with the characteristic pattern of C-starvation conditions. This toxicity response resulted in the redistribution of N from amino acids, mostly asparagine, and lower C/N ratios. The C/N imbalance at high NH(4)(+) concentration under control conditions induced a strong activation of root C metabolism and the upregulation of anaplerotic enzymes to provide C intermediates for the tricarboxylic acid cycle. A high light intensity partially reverted these C-starvation symptoms by providing higher C availability to the plants. The extra-C contributed to a lower C4/C5 amino acid ratio while maintaining the relative contents of some minor amino acids involved in key pathways regulating the C/N status of the plants unchanged. C availability can therefore be considered to be a determinant factor in the tolerance/sensitivity mechanisms to NH(4)(+) nutrition in plants.

High irradiance increases NH4 + tolerance in Pisum sativum: Higher carbon and energy availability improve ion balance but not N assimilation

The widespread use of NO(3)(-) fertilization has had a major ecological impact. NH(4)(+) nutrition may help to reduce this impact, although high NH(4)(+) concentrations are toxic for most plants. The underlying tolerance mechanisms are not yet fully understood, although they are thought to include the limitation of C, the disruption of ion homeostasis, and a wasteful NH(4)(+) influx/efflux cycle that carries an extra energetic cost for root cells. In this study, high irradiance (HI) was found to induce a notable tolerance to NH(4)(+) in the range 2.5-10mM in pea plants by inducing higher C availability, as shown by carbohydrate content. This capacity was accompanied by a general lower relative N content, indicating that tolerance is not achieved through higher net N assimilation on C-skeletons, and it was also not attributable to increased GS content or activity in roots or leaves. Moreover, HI plants showed higher ATP content and respiration rates. This extra energy availability is related to the internal NH(4)(+) content regulation (probably NH(4)(+) influx/efflux) and to an improvement of the cell ionic balance. The limited C availability at lower irradiance (LI) and high NH(4)(+) resulted in a series of metabolic imbalances, as reflected in a much higher organic acid content, thereby suggesting that the origin of the toxicity in plants cultured at high NH(4)(+) and LI is related to their inability to avoid large-scale accumulation of the NH(4)(+) ion.

Sf29 Gene of Spodoptera frugiperda Multiple Nucleopolyhedrovirus Is a Viral Factor That Determines the Number of Virions in Occlusion Bodies

ABSTRACT The genome of Spodoptera frugiperda multiple nucleopolyhedrovirus (NPV) was inserted into a bacmid (Sfbac) and used to produce a mutant lacking open reading frame 29 (Sf29null). Sf29null bacmid DNA was able to generate an infection in S. frugiperda. Approximately six times less DNA was present in occlusion bodies (OBs) produced by the Sf29null bacmid in comparison to viruses containing this gene. This reduction in DNA content was consistent with fewer virus particles being packaged within Sf29null bacmid OBs, as determined by fractionation of dissolved polyhedra and comparison of occlusion-derived virus (ODV) infectivity in cell culture. DNA from Sfbac, Sf29null, or Sf29null-repair, in which the gene deletion had been repaired, were equally infectious when used to transfect S. frugiperda. All three viruses produced similar numbers of OBs, although those from Sf29null were 10-fold less infectious than viruses with the gene. Insects infected with Sf29null bacmid died ∼24 h later than positive controls, consistent with the reduced virus particle content of Sf29null OBs. Transcripts from Sf29 were detected in infected insects 12 h prior to those from the polyhedrin gene. Homologs to Sf29 were present in other group II NPVs, and similar sequences were present in entomopoxviruses. Analysis of the Sf29 predicted protein sequence revealed signal peptide and transmembrane domains, but the presence of 12 potential N-glycosylation sites suggest that it is not an ODV envelope protein. Other motifs, including zinc-binding and threonine-rich regions, suggest degradation and adhesion functions. We conclude that Sf29 is a viral factor that determines the number of ODVs occluded in each OB.

Two Fe-superoxide dismutase families respond differently to stress and senescence in legumes.

Three main families of SODs in plants may be distinguished according to the metal in the active center: CuZnSODs, MnSOD, and FeSOD. CuZnSODs have two sub-families localized either in plant cell cytosol or in plastids, the MnSOD family is essentially restricted to mitochondria, and the FeSOD enzyme family has been typically localized into the plastid. Here, we describe, based on a phylogenetic tree and experimental data, the existence of two FeSOD sub-families: a plastidial localized sub-family that is universal to plants, and a cytosolic localized FeSOD sub-family observed in determinate-forming nodule legumes. Anti-cytosolic FeSOD (cyt_FeSOD) antibodies were employed, together with a novel antibody raised against plastidial FeSOD (p_FeSOD). Stress conditions, such as nitrate excess or drought, markedly increased cyt_FeSOD contents in soybean tissues. Also, cyt_FeSOD content and activity increased with age in both soybean and cowpea plants, while the cyt_CuZnSOD isozyme was predominant during early stages. p_FeSOD in leaves decreased with most of the stresses applied, but this isozyme markedly increased with abscisic acid in roots. The great differences observed for p_FeSOD and cyt_FeSOD contents in response to stress and aging in plant tissues reveal distinct functionality and confirm the existence of two immunologically differentiated FeSOD sub-families. The in-gel FeSOD activity patterns showed a good correlation to cyt_FeSOD contents but not to those of p_FeSOD. This indicates that cyt_FeSOD is the main active FeSOD in soybean and cowpea tissues. The diversity of functions associated with the complexity of FeSOD isoenzymes depending of the location is discussed.

Expression and Localization of a Rhizobium-Derived Cambialistic Superoxide Dismutase in Pea (Pisum sativum) Nodules Subjected to Oxidative Stress

Two phylogenetically unrelated superoxide dismutase (SOD) families, i.e., CuZnSOD (copper and zinc SOD) and FeMn-CamSOD (iron, manganese, or cambialistic SOD), eliminate superoxide radicals in different locations within the plant cell. CuZnSOD are located within the cytosol and plastids, while the second family of SOD, which are considered to be of bacterial origin, are usually located within organelles, such as mitochondria. We have used the reactive oxygen species-producer methylviologen (MV) to study SOD isozymes in the indeterminate nodules on pea (Pisum sativum). MV caused severe effects on nodule physiology and structure and also resulted in an increase in SOD activity. Purification and N-terminal analysis identified CamSOD from the Rhizobium leguminosarum endosymbiont as one of the most active SOD in response to the oxidative stress. Fractionation of cell extracts and immunogold labeling confirmed that the CamSOD was present in both the bacteroids and the cytosol (including the nuclei, plastids, and mitochondria) of the N-fixing cells, and also within the uninfected cortical and interstitial cells. These findings, together with previous reports of the occurrence of FeSOD in determinate nodules, indicate that FeMnCamSOD have specific functions in legumes, some of which may be related to signaling between plant and bacterial symbionts, but the occurrence of one or more particular isozymes depends upon the nodule type.

Regenerable Plasmonic Biosensor Based on Gold Nanolines Pattern and Common Laboratory Spectrophotometer

Gold nanostructures can undergo plasmonic behavior without need of light couplers like in traditional surface plasmon resonance (SPR) systems. This effect, known as LSPR (localized SPR), can be exploited to develop optical biosensors in simple configuration. In this paper, an LSPR system based on gold lines nanopattern as transducer has been developed by using a common laboratory spectrophotometer as reader and an adapted flow cell. This novel system was applied to anti-immunoglobulin G (IgG) detection, as proof of concept. For this purpose, the transducer surface was covalently biofunctionalized with IgG and incubated with increasing concentrations of anti-IgG. The response was determined by measuring changes in light transmission spectra (Δλ, in nanometers). Contrary to other reported nanopatterns, biosensor features as repeatability, regeneration, and detectability have been deeply studied. Results indicate that the system can act as a robust and regenerable biosensor with a limit of detection of 5.98 ± 1.34 nM.

Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) technique is based on the specific recognition ability of the molecular structure of an antigen (epitope) by an antibody and is likely the most important diagnostic technique used today in bioscience. With this methodology, it is possible to diagnose illness, allergies, alimentary fraud, and even to detect small molecules such as toxins, pesticides, heavy metals, etc. For this reason, any procedures that improve the detection limit, sensitivity or reduce the analysis time could have an important impact in several fields. In this respect, many methods have been developed for improving the technique, ranging from fluorescence substrates to methods for increasing the number of enzyme molecules involved in the detection such as the biotin–streptavidin method. In this context, nanotechnology has offered a significant number of proposed solutions, mainly based on the functionalization of nanoparticles from gold to carbon which could be used as antibody carriers as well as reporter enzymes like peroxidase. However, few works have focused on the study of best practices for nanoparticle functionalization for ELISA enhancement. In this work, we use 20 nm gold nanoparticles (AuNPs) as a vehicle for secondary antibodies and peroxidase (HRP). The design of experiments technique (DOE) and four different methods for biomolecule loading were compared using a rabbit IgG/goat anti-rabbit IgG ELISA model (adsorption, directional, covalent and a combination thereof). As a result, AuNP probes prepared by direct adsorption were the most effective method. AuNPs probes were then used to detect gliadin, one of the main components of wheat gluten, the protein composite that causes celiac disease. With this optimized approach, our data showed a sensitivity increase of at least five times and a lower detection limit with respect to a standard ELISA of at least three times. Additionally, the assay time was remarkably decreased.

Conjugation of Active Iron Superoxide Dismutase to Nanopatterned Surfaces

Superoxide dismutase enzymes (SODs) are an essential part of the first line of cellular defense system against free radicals species. They catalyze the dismutation of superoxide radicals into oxygen and hydrogen peroxide. Although several studies have examined the attachment of superoxide dismutases to nanoparticles and nanostructures, never has been used a member of the Fe/MnSOD family. In this study, the behavior of plant origin FeSOD enzyme on three different nanopatterned surfaces was investigated as a function of covalent and electrostatic binding. Fluorescence microscopy was used to demonstrate that the protein is attached only to the gold layer. We also examined the activity of SOD by a colorimetric assay, and we have shown that the enzyme remains active after attachment to the three different surfaces under both kind of binding (electrostatic and covalent). This methodology could be useful for those who want to functionalize nanostructures with a SOD enzyme and test the activity. This process could be of great interest for the development of peroxynitrite and superoxide biosensors.

LSPR Cuvette for Real-Time Biosensing by Using a Common Spectrophotometer

Although localized surface plasmon resonance (LSPR) transducers can be optically interrogated without the need of light couplers, normally they need tailor-made optical assemblies for signal monitoring. This fact limits the application of LSPR biosensors in laboratories due to lack of specific optical setups. If commercial equipment like common spectrophotometers were suitable for LSPR interrogation, the LSPR technology could be applied more often in detection applications. In this paper, a universal LSPR cuvette has been developed in order to use a commercial spectrophotometer as a real-time biosensor without the need of any extra optical component. By measuring the changes in absorbance at constant wavelength, the equipment provides sensograms where real-time response is monitored. The cuvette, fully compatible with spectrophotometers, is an LSPR flow-cell (volume less than 10 mL) where a gold nanopatterned surface is emplaced as a window. Gold nanopatterns (period 600 nm) based on lines and pillars were simulated, fabricated, measured, and compared. The resolutions of

Direct electrochemistry and environmental sensing of rice hemoglobin immobilized at graphite electrodes

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