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Protective role of antioxidant enzymes in legume nitrogen fixation


Research objectives and content
Nitrogen fixation is a process that needs to be promoted under natural conditions because it is is largely beneficial, both economically and environmentally, as compared with chemical fertilization. Legwne root nodules contain a variety of antioxidant metabolites and enzymes to cope with activated oxygen species formed in the respiration process. Two critical antioxidant enzymes are ascorbate peroxidase (APX) and gluthatione reductase (GR). Lipoxygenase (LOX) is a likely candidate to generate activated oxygen because it catalyses the oxidation of polyunsaturated fatty acids. By studying simultaneouslythe expression of antioxidant enzymes and nitrogenase during nodule ontogeny and senescence, important information can be gained on the protective role of antioxidants during nodule operation and on the mechanism underlying senescence. I will investigate in detail APX, GR, nitrogenase and LOX. My main objectives will be to study how expression of APX and GR is regulated during nodule forrnation and senescence, and to demonstrate that these antioxidants are involved in the protection of nitrogenase by using a combination of molecular, cellular, and physiological techniques. As a summary, the particular tasks to be performed within the above-mentioned objectives are the measurement of enzyme activities (spectrophotometly, fluorometry, gas flow-through systems), proteins (Western analysis, immunolocalization) and mRNAs (Northern analysis, in situ hybridization) for APX, GR, nitrogenase and LOX. Details are given on the research description.
Training content (objective, benefit and expected impact)
This type of approach is novel and should yield important results with application in agriculture. It will provide irnportant inforrnation and some clues as to why nitrogen fixation is limited during nodule senescence, both natural and s1ress induced. It will allow me also to learn the most modem techniques of enzyme and transcript localization and to receive training in studies of structure-function relationship in legume-nodules. My stay will be also beneficial for the groups mvolved in my forrnation, will consolidate the established collaborations between the Spanish and UK groups, and will allow me to continue my research, from a wider scientific and more European geographical perspective, upon my return to Spain. The information obtained is essential to identify critical antioxidants and their mechanisms of action in vivo if antioxidant levels are to be manipulated using transgenic legumes of agronomic interest.
Links with industry / industrial relevance (22)
Lucerne is increasingly being used as an environmentally-friendly crop in Spain and other European countries (it lacks the problems associated with nitrate pollution and improves landscaping), and as a prime protein source for livestock. It is also a crop promoted by the Common Agriculture Policy (CAP) of the EC. As many as 57 industries are directly dependant on lucerne production in Spain. This proposal is supported by the 'Spanish Association for the Research and Improvement of Luceme', whose official signature is provided in an accompanying letter.


John Innes Centre
Sainsbury Laboratory Colney Lane
NR4 7UH Norwich
United Kingdom