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Final Report Summary - AMUSE (Identification of new molecular and genetic basis of ammonium use efficiency in plants)

The CIG project “Identification of new molecular and genetic basis of ammonium use efficiency in plants (AMUSE)” has allowed me to start and independent research line integrated in the Nutrition Management in Plant and Soil (NUMAPS) research group of the University of the Basque Country UPV/EHU and to contribute to European research in the area of plants nitrogen nutrition.
Global context of the project

One of the major challenges that smart agriculture needs to face is sustainability. This means to maintain equilibrium between obtaining a product at the best possible yield and with the best possible quality at the same time that the minimum environmental impact is provoked. Excessive nitrogen (N) fertilization is one of the main aspects to be improved in agriculture because of nitrate (NO3−) leaching that gives rise to soil and water pollution. Moreover, incomplete capture and poor conversion of N fertilizers also cause global warming, mostly through emissions of nitrous oxide (N2O). Besides, NO3- can also accumulate in plants, especially in leafy vegetables, being harmful for human health. In this context, the use nitrification inhibitors together with ammonium (NH4+) based fertilizers has been proven useful to mitigate the undesirable effects of nitrogen-fertilization. Thus, the potential of NH4+ as N source for agriculture has been reconsidered alongside the search to improve N use efficiency by the Intergovernmental Panel on Climate Change (IPCC).

Although NH4+ is a fundamental substrate for biomolecules, when present in excess most plant species develop toxic symptoms, being more acute when NH4+ is provided as the sole N source. However, there exists great intra ant interspecific variability in ammonium tolerance or sensitivity. In this sense, rice or coniferous trees are considered as tolerant species while most agricultural crops are considered as sensitive species. Ammonium toxicity syndrome in plants includes, among others, leaf chlorosis, ion imbalance, disorder in pH regulation and changes in metabolites levels, which finally derive in poor biomass accumulation of NH4+-fed plants compared with NO3–-fed ones. Nevertheless, it has been observed that the metabolic adaptation to NH4+ may also have benefits. In terms of food quality, a frequent characteristic of NH4+-fed plants is an increase in protein content, which is commonly associated with the need to increase NH4+ assimilation in order to prevent its accumulation. Although in recent years important advances have been done, the reasons underlying ammonium toxicity are not still completely understood and surprisingly the molecular components involved in ammonium stress are still barely known. In this context, the main objective of the project was to further understand plants response upon ammonium stress and to advance in the characterization of the molecular and genetic basis of ammonium use efficiency.

Work performed and main results

We firstly engaged a natural variation study with 47 natural accessions of Arabidopsis thaliana and we observed high intraspecific variability of ammonium tolerance expressed as shoot biomass ratio of ammonium-fed versus nitrate-fed plants. We found that tissue ammonium accumulation was an important determinant of the natural variability in shoot biomass independently of the N source provided (Sarasketa et al., 2014 Journal of Experimental Botany). We are at present continuing to use natural variation studies to find actors involved in Arabidopsis differential growth in function of the N source.

Besides, we established hydroponic axenic conditions for studying Arabidopsis thaliana to growing with NO3- or NH4+ as N source and we observed the effect of varying N concentration (2 and 10 mM) and medium pH (5.7 and 6.7) on plant performance. The results, published in Sarasketa et al. (2016, Frontiers in Plant Science), evidenced changes in the response of ammonium assimilation machinery and of the anaplerotic enzymes associated to Tricarboxylic Acids (TCA) cycle in function of the plant organ, the N source and the degree of ammonium stress. A greater stress severity at pH5.7 was related to NH4+ accumulation and could not be circumvented in spite of the stimulation of glutamine synthetase, glutamate dehydrogenase, and TCA cycle anaplerotic enzymes.

Once established these growth conditions, we chose a situation where plants displayed ammonium tolerance, 2 mM concentration and pH 6.7, to perform an “omics” approach with the aim to identify molecular players involved in Arabidopsis growth under different N sources. A quantitative proteomic approach, published in Marino et al., 2016 (Journal of Experimental Botany) evidenced that glucosinolate metabolism was differentially regulated by the N source, because TGG1 and TGG2 myrosinases were more abundant under ammonium nutrition. Indeed, Arabidopsis plants displayed glucosinolate accumulation and induced myrosinase activity under ammonium nutrition. Moreover, these metabolic changes were correlated in Arabidopsis with the differential expression of genes from the aliphatic glucosinolate metabolic pathway.

Besides, the work performed in Arabidopsis thaliana model plant we have also worked in economically important crops. In Brassica oleracea (broccoli) leaves we confirmed the results found in Arabidopsis regarding glucosinolate metabolism interaction with the nitrogen source (Marino et al., 2016 Journal of Experimental Botany). More recently we also performed experiments in Brassica napus (rapeseed) focused in the interaction between N source and sulfur metabolism with special attention to glucosinolate metabolism. This work has already been submitted for publication. The overall results results underline the importance of N nutrition and highlight the potential of using NH4+ as the N source in order to stimulate glucosinolate metabolism, which may have important applications not only in terms of reducing pesticide use, but also for increasing plants’ nutritional value.

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Life Sciences
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