Name:N_limitation_N2_aerial - growth conditions - nitrogen,nitrogen:time 4day . The nodulated root system of six plants is split into two parts. The major root part is supplied with nitrogen-free nutrient solution (N2 is replaced by argon) whereas the minor part remain on air. The nutrient solutions are renewed daily. After four days of this treatement of N-limitation, the shoots are individually collected, weighted, immediately frozen and stored at minus 80 degre Celsius.
Growth protocol
root - Plants were grown hydroponically. Plants were cultivated in growth chamber with the following environmental parameters: light/dark cycle 8/16h, light intensity 250 umol.s-1.m-2 PAR, temperature 22degreeC/20degreeC, 70% hygrometry. The nutrient solution was renewed twice a week during the first part of the culture, and daily the last week before the experiment. Plants were grown during two weeks in a nutrient solution contained 1 mM KNO3, 1 mM KH2PO4 , 1 mM MgSO4 , 250 uM CaCl2 , 0.1 mM Na-Fe-EDTA, 50 uM KCl, 50 uM H3BO3, 5uM MnSO4, 1 uM ZnSO4, 1 uM CuSO4, 0.1 uM Na6Mo7O24, pH adjusted to 6 with KOH. After these two weeks, plants were transferred in the same nutrient solution devoid of nitrogen, adjusted to pH7 and they are inoculated with Sinorhizobium meliloti strain 2011. Strong aeration of nutrient solution is achieved by blowing air with an aquarium pump in pumice stone diffuser. Two weeks after inoculation, nodulated plants were transferred on aerated nitrogen free nutrient solution, adjusted to pH6.
Experiment has been designed to characterize the molecular expression patterns associated to a contrasted modification of the nitrogen status of the whole plant. The systemic effects of nitrogen status modifications are investigated and compared on non nodulated plant supplied with NO3, NH4 or nodulated plants (Sinorhizobium meliloti 2011) supplied with air. The root systems were separated in two compartments of unequal sizes (split root system). Two treatments were applied on the larger compartment in order to modulate the nitrogen status of the plant: for the S treatment, roots are supplied with nutrient solution containing 10 mM NH4NO3,, whereas for the C treatment, roots are supplied with nitrogen free medium. In the case of N2 fixing plants, N limitation was obtained by replacing air by a mixture of Ar and O2 80 per cent and 20 per cent. The effects of these treatments were investigated on roots of the minor compartment supplied continuously with either NO3 1 mM, NH4 1 mM or air (N2) and on the shoots. We were also interested in the molecular expression patterns associated to the roots deprived of N.
Data processing
The data were normalized with the gcrma algorithm (Irizarry et al., 2003), available in the Bioconductor package (Gentleman and Carey, 2002).
