NITRATE METABOLISM IN LETTUCE AND ROCKET

Abstract Intensive farming techniques with highly fertilization management lead to produce leafy vegetables with high accumulation of nitrates. The Reg. UE 1258/2011 imposes threshold on nitrate content beyond which the vegetables (lettuce, rocket and spinach) cannot be placed on the market. It is so important to adopt strategies which reduce nitrate levels in leaves. This objective can be achieved through the studies on the metabolism of nitrates in model and greatest commercial interest species. This study focuses on cultivation in floating system because was shown that this cultivation technique has the ability to reduce the intake of macronutrients in the nutrient solution, making more efficient the assimilation of nitrogen by the plants and so reducing the level of nitrates in vegetables (Rouphael et al., 2004). Two leafy vegetables were chosen as study-species, lettuce (Lactuca sativa L.) and rocket (Diplotaxis tenuifolia L.) that have different efficiency of use and organication of nitrates. Some researchers have found levels of nitrate in Diplotaxis higher than 9300 mg*kg-1 FW (Santamaria et al., 1999; Cerutti et al., 1996), in fact rocket it is considerate a hyper accumulator of nitrates. This research focuses on comparative biology studies between the two species and on their nitrate metabolism to understand which are the factors that make the difference in the nitrate accumulation. Nitrate reductase, NR, (E.C.1.7.1.1-3) is the first enzyme from which start NO3- organication. Concentration in nitrates in the nutrient solution and other abiotic factors like light intensity and exposure, diurnal alternation of light and dark, temperature, CO2 levels, hormones (cytokinin, ethylene) (Dordas, 2009), anoxia, availability of sugars and nitrogen metabolites such as glutamine all play regulatory roles in NR activity (Crawford, 1995). The growing experiments performed for this thesis were planned to characterize the nitrate metabolism in lettuce and rocket plants grown in nutrient solutions containing different nitrate concentrations and under different light exposure. To achieve this objective the plants were cultivated, on the one hand, in conditions very similar to reals one, in greenhouse and with nutrient solution containing 2, 10 and 20 mM NO3-, but also with nutrient solution with low nitrate concentrations, 0.25, 0.5, 1 and 2 mM NO3-, gave after 1 day of nitrogen starvation, to highlight the high sensitiveness of nitrate transporters, and consequently on nitrate reductase, at small differences of nitrate in the nutrient solution. On the other hand the cultivations were carried out in a growth chamber in order to eliminate the influence on the results of some environmental parameters, which are difficult to evaluate in an uncontrolled environment and the aforesaid low nitrate concentrations were tested. The qualitative parameters, as content of chlorophylls, carotenoids, nitrates, nitrites, sucrose, reducing and total sugars were determinate to understand the status of the plants sampled at different environmental conditions and to evaluate how the different concentration of nitrates in the nutrient solutions affect these parameters. Then the activity of NR was measured using two different assays to understand the response of this enzyme at the different conditions used and to be correlated with the qualitative parameters, in order to deepen the mechanisms that affect the first step of reduction of the nitrates and, consequently, their organication to glutamate and amino acids. The study was integrated with the gene expression analysis for the main enzymes involved in the nitrate metabolism of lettuce and rocket: nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase and nitrate transporter (NTR). These analysis were performed using quantitative retro-transcriptase PCR (qRT-PCR) to study the transcriptional regulation under the different nitrate concentrations availability.