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 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.
Try out step step step step 3 (amino acid 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 picking, nitrogen and isotope analysis
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 CaSO4 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 N content from plant samples was in fact measured in continuing move having fun with a-c/N analyzer linked to a keen isotope ratio bulk spectrometer (Roboprep CN and you will 20–20 size spectrometer; Europa PDZ, Crewe, UK).
Phloem exudates have been compiled utilizing the triggerred diffusion strategy by the EDTA considering King & Zeevaart (1974 ) and you will Bourgis ainsi que al. (1999 ). Plant stems was indeed completely clipped on crown peak having a shaver blade inside the EDTA provider (20 meters yards , pH eight.0). The typical diameter of your own stalk areas was about 6 mm. Excision sections had been after that rinsed in the a separate EDTA provider (5 m yards , pH 7.0) for five minute and listed lesbian dating site in Canada in vials who has 2 mL (N-starvation experiment) or 8 mL (gains duration check out) of the same services. Exudation is actually triggered at nighttime inside the a beneficial climatized chamber at 20 °C having a water-over loaded surroundings (Hours = 90%). This new exudation provider (5 yards yards EDTA, pH 7.0) was revived most of the 2 h inside collection age several h. The newest EDTA of every compiled fraction is actually precipitated by the addition of a hundred µL of 0.5 N HCl and you may stored for 1 h during the ?20 °C. The newest products had been upcoming held overnight at the 4 °C in advance of centrifugation at the 5000 grams to own 10 minute. The brand new supernatants had been modified so you can pH 5.9, filtered owing to a beneficial 0.45 µm nylon membrane layer and held on ?20 °C prior to amino acid analyses. 100 % free amino acids from inside the phloem exudates had been analysed by highest-abilities liquids chromatography (HPLC) since ophtaldialdehyde derivatives into a c-18 column using good 32 Karat Program (Beckman Products, San Ramon, Ca, USA) since the in past times discussed from the Murray, Hatch & Cliquet (1996 ). Particular proteins was quantified using ?-aminobutyric acidic as the an internal basic.