Plants from the field were harvested at the Cdos stage, screened for their tap root diameter (0.6–1.0 cm), before being acclimated in a climate-controlled room under local hookups in Pueblo hydroponic culture conditions as described in the previous section. Developmental stages were estimated from the phenological calendar established by the Bayer, BASF, Ciba and Hoechst companies. Nine plants for each developmental stage of the growth cycle were used for measuring HATS influx and BnNrt2 mRNA abundance. Six other plants were used for assaying amino acids content in phloem exudates.
Test 3 (amino acidic consequences)
Six pots of 25 plants were first transferred to N-free nutrient solution for 12 h before being transferred to a new nutrient solution containing 1 m m KNO3 and either 100 µ m GABA, 1 m m GABA, 1 m m Gln, 1 m m Glu or 1 m m Asn for 12 h. At the end of the treatments, HATS influx, BnNrt2 mRNA abundance and root amino acids content were measured on six, two and three replicates, respectively.
Plant harvesting, nitrogen and you can isotope studies
In order to measure nitrate influx, plants of the three experiments were grown in hydroponic tanks and were treated according to the particular experimental design. Roots were then washed twice in 1 m m CaSOcuatro solution for 1 minute at 20 °C to remove unabsorbed K 14 NO3 and transferred to influx solution containing 100 µ m of K 15 NO3 (99.9%) for 5 min. Plants were then washed twice in 1 m m CaSO4 solution for 1 min at 4 °C to slow down the metabolism and remove unabsorbed tracer residing in the cell wall space. Roots and shoots were harvested separately and dried for 48 h at 60 °C before being ground to fine powder for isotope analysis. A root fraction of each plant was frozen in liquid nitrogen and stored at ?80 °C for amino acids and northern blot analyses.
Nitrogen and you may fifteen Letter stuff away from plant samples was in fact measured in persisted move playing with a c/Letter analyzer related to an isotope ratio size spectrometer (Roboprep CN and 20–20 size spectrometer; Europa PDZ, Crewe, UK).
Phloem exudates was basically accumulated using the triggerred diffusion means from the EDTA according to Queen & Zeevaart (1974 ) and you may Bourgis et al. (1999 ). Bush stems was in fact completely cut within crown height with an excellent razor blade inside the EDTA provider (20 m meters , pH seven.0). An average diameter of the stem parts involved 6 mm. Excision parts have been next rinsed into the a unique EDTA service (5 m m , pH 7.0) for five minute and you will listed in vials that features 2 mL (N-deprivation try out) otherwise 8 mL (gains years experiment) of the same provider. Exudation is actually induced at night in an effective climatized chamber during the 20 °C having a h2o-over loaded environment (Time = 90%). Brand new exudation solution (5 yards yards EDTA, pH eight.0) was renewed every 2 h for the collection age of several h. The fresh new EDTA of each and every obtained small fraction try precipitated by adding a hundred µL off 0.5 Letter HCl and held for one h within ?20 °C. The latest products was in fact upcoming kept instantly at the 4 °C ahead of centrifugation within 5000 g to have 10 minute. The latest supernatants have been adjusted so you can pH 5.nine, blocked courtesy a great 0.forty-five µm nylon membrane and held during the ?20 °C prior to amino acidic analyses. Free amino acids from inside the phloem exudates had been analysed because of the higher-performance water chromatography (HPLC) since the ophtaldialdehyde derivatives to the a c-18 line having fun with a good 32 Karat Program (Beckman Tool, San Ramon, California, USA) because the previously revealed by Murray, Hatch & Cliquet (1996 ). Particular proteins was quantified playing with ?-aminobutyric acid since the an interior basic.