Research Areas

• Nitrogen nutrition in plants: nitric, ammonium and ureic nutrition and atmospheric fixation by leguminous plants
• Abiotic stress as yield limiting factor: Physiological mechanisms of vegetable antioxidants
• Ammonium stress and tolerance mechanisms (in pea , spinach, and arabidopsis plants as models)
• Biotechnological and nanotechnological use of Plants’ Antioxidant Systems

Nitrogen nutrition in plants: nitric, ammoniacal and ureic nutrition and atmospheric fixation by leguminous plants

Diffuse contamination from agriculture is now one of the largest environmental problems faced by many European countries. The greatest concern surrounds two issues: firstly, the increased concentration of nitrates in surface and underground waters, and, secondly, atmospheric pollution in augmenting the greenhouse effect, the destruction of the ozone layer and acid rain. These environmental and sanitary problems produced by the use of nitrogen fertiliser in agriculture have led to a compromise between the obtainment of high yields and quality crops, and minimal environmental impact. In order to achieve “sustainable” agricultural production, it is necessary to study in depth the processes related to the transformations of nitrogen in the soil and the different ways in which N is absorbed by plants and how these affect their growth and development. On the other hand it will be also necessary to develop new fertilisers and forms of application associated with the use of products capable of inhibiting the activities of those microorganisms involved in the transformations of nitrogen present in the soil. Consequently, the more we know about integral plant physiology (not just the physiological and biochemical aspects responsible for N assimilation, but their interaction with the metabolism of carbon), the more capable we shall be of progressing in the whole process of plant production. This knowledge may include the design of new types of fertilisers to reduce the environmental impact resulting from the use of nitrogen fertilisers, or the basics for plant breeding either by plant transformation or by using traditional methods to obtain new varieties which exhibit the metabolic mechanisms needed to attain greater efficiency in the use of nitrogen

Abiotic stress as yield limiting factor: Physiological antioxidant defence mechanisms in plants

Abiotic stress is the main limiting factor affecting vegetable crop yields, proving more significant than the limitations caused by nutrients and biotic stress (pathogens and pests). The selection of modern varieties and the pressure of agricultural farming is subjected to mean that certain environmental stress factors have become one of the main issues which needs to be tackled. In Spain, drought, cold weather, high temperatures, soil-associated nutritional deficiencies, the salinity of certain terrains, excessive solar irradiance in some areas and other types of stress, or even a combination of these, often lead to vegetable senescence and greatly reduce not only vegetable yields, but also quality, producing incertitude in the minds of end farmers when it comes to making decisions. Many of these stresses are capable of upsetting the balance between the production of free radicals and active oxygen species (AOS), and plants’ antioxidant defences, a situation which implies an increase in oxidative damage to cellular components and oxidative stress. The first work to demonstrate that abiotic stress, such as drought, could lead to oxidative stress was performed with pea plants (Moran et al., 1994).

Ammonium stress and tolerance mechanisms (in pea, spinach and arabidopsis plants as models)

Tolerance to ammonium nutrition implies a great demand for carbon skeletons for the assimilation of ammonium in the roots, giving rise to the accumulation and mistargeting of amino compounds (amino acids, polyamines, urea). The high availability of amino acids may accelerate the release of photorespiratory ammonium and its emission into the atmosphere in the form of ammonium. High levels of glutamate favour the synthesis of ornithine and citrulline, precursors of arginine. The urea cycle is the connection point between these metabolites. The coordinated action of arginase and urease could provide a mechanism to recycle urea N or encourage the volatilisation of ammonium. Knowledge of the balance between the emission and consumption of N in soil and plants is essential when it comes to defining sustainable fertilisation policies.

Biotechnological and nanotechnological use of Plants’ Antioxidant Systems

Leguminous nodules are a good model to study plants' antioxidant systems with. Due to the lability of the components of nitrogenase, the enzymatic complex which biologically fixes atmospheric nitrogen, defence systems against AOS are required. The synthesis of leghaemoglobins and O2 diffusion barriers are a normal defence systems in leguminous nodules. Other leguminous antioxidants also present include superoxide dimutase (SOD), ascorbate-glutathione cycle enzymes and low-molecular-mass antioxidants, such as ascorbate, glutathione and homoglutathione, present in high concentrations in the nodules (Dalton 1995). There is little doubt that the presence of all these antioxidants categorically reveals the problems of the nitrogen fixation process with oxygen and its AOS.

New techniques of DNA cloning and recombinant protein expression may permit the large-scale overexpression of proteins of vegetable origin with antioxidant functions in prokaryotic organisms [Moran et al., (2002; 2003)] or eukaryotic organisms [Iturbe-Ormaetxe et al., (1992)]. The overexpression in these heterologous biological systems allows large quantities of proteins (not highly abundant in the cell of origin) to be obtained, making it possible to learn about and characterise these proteins of plant origin with antioxidant functions. Of these, we have participated in the characterisation of the following: Cowpea leghaemoglobin II, the first recombinant haemoglobin capable of binding O2 reversibly (both in plants and humans) [Arredondo-Peter et al., (1997)]; Rice haemoglobin I, a non-symbiotic haemoglobin which possesses the greatest affinity for oxygen known to date [Arredondo-Peter et al., (1997b)]; Ferric leghaemoglobin reductase from soybean nodules, an enzyme which reduces Lb3+ to its functional, physiological form, Lb2+O2 [Moran et al., (2002); Urarte et al (2008)]; and FeSOD from cowpea nodules [Moran et al., (2003)], against which monospecific antibodies have been obtained and which is the first FeSOD from eukaryotes whose 3-D structure has been described [Muñoz et al., (2003; 2005).

Leguminous nodules are also an interesting model system for the study of haemoglobins and antioxidants; of these, we shall focus on SOD. SOD is an antioxidant enzyme essential to oxygenic life. It is, however, easily inactivated by nitrative attack (via the peroxynitrite anion, a potent oxidant). This fact has been used in a recent patent to estimate the oxidative damage to recombinant FeSOD caused by peroxynitrite in an in vitro bioassay (Moran et al. Patent No. 200702035).