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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.
Test step three (amino acid effects)
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 will isotope investigation
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 15 N posts out of plant products was in fact counted inside the persisted disperse playing with a c/Letter analyzer associated with a keen isotope proportion mass spectrometer (Roboprep CN and 20–20 bulk spectrometer; Europa PDZ, Crewe, UK).
Phloem exudates were compiled with the triggerred diffusion approach by the EDTA predicated on Queen & Zeevaart (1974 ) and you will Bourgis ainsi que al. (1999 ). Bush stems was basically completely slashed at the top level that have a beneficial razor blade within the EDTA solution (20 m m , pH seven.0). The average diameter of your own stalk areas involved 6 mm. Excision areas was indeed upcoming rinsed into the an alternative EDTA services (5 yards m , pH eight.0) for 5 minute and you will https://datingranking.net/local-hookup/ventura/ listed in vials which has 2 mL (N-deprivation check out) or 8 mL (development duration test) of the same services. Exudation is created at nighttime within the an effective climatized chamber on 20 °C that have a liquid-over loaded atmosphere (Hr = 90%). The brand new exudation services (5 m m EDTA, pH 7.0) try renewed all of the 2 h during the collection period of twelve h. The latest EDTA of each built-up tiny fraction was precipitated by adding 100 µL regarding 0.5 N HCl and you may stored for 1 h within ?20 °C. The newest samples was basically up coming held instantly in the 4 °C in advance of centrifugation from the 5000 grams to own 10 minute. The newest supernatants had been adjusted in order to pH 5.nine, filtered by way of good 0.forty five µm plastic membrane and held at ?20 °C ahead of amino acid analyses. Totally free proteins during the phloem exudates was basically analysed because of the high-abilities h2o chromatography (HPLC) due to the fact ophtaldialdehyde derivatives to your a-c-18 line playing with an excellent 32 Karat Program (Beckman Tool, San Ramon, California, USA) given that in the past demonstrated of the Murray, Hatch & Cliquet (1996 ). Particular proteins have been quantified using ?-aminobutyric acid since the an internal fundamental.