TITLE: Root nitrate influx 
AUTHORS: Prajakta Bendre, Vanessa Melino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><pre><code><div class = "text-blocks"></div></code></pre></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Nitrogen (N) in the form of nitrate (NO</span><span style = "vertical-align:sub;vertical-align:super;">3</span><span style = "vertical-align:sub;vertical-align:super;">-</span><span>) can move radially from roots to the vascular tissue from where it is transported to the above-ground organs. Roots are the first to sense the scarcity of nitrogen in the soil. The following protocol describes the step-wise procedure for performing unidirectional root NO</span><span style = "vertical-align:sub;vertical-align:super;">3</span><span style = "vertical-align:sub;vertical-align:super;">-</span><span> influx with the use of </span><span style = "vertical-align:super;">15</span><span>N labelled NO</span><span style = "vertical-align:sub;vertical-align:super;">3</span><span style = "vertical-align:sub;vertical-align:super;">-</span><span> at a concentration that stimulates the inducible high-affinity nitrate transporters. Stable isotopes such as </span><span style = "vertical-align:super;">15</span><span>N can be used to trace the movement of NO</span><span style = "vertical-align:sub;vertical-align:super;">3</span><span style = "vertical-align:sub;vertical-align:super;">-</span><span> through the plant. This protocol uses a split-root design where only embryonic roots are treated with the </span><span style = "vertical-align:super;">15</span><span>N label. The remaining, non-embryonic roots are not labelled but similarly treated. this split-root design can be modified to select and label specific root types based on your interest.</span></div><style>
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[STEPS]
?. Grow barley plants hydroponically according to the desired growth conditions.
?. Prepare the set up by pre-filling 50 ml centrifuge tubes with solutions (1-4 above) and aerate.
?. [DAY 18]
18- day old barley plants were removed from their collars and two plants were bundled together at the stem. These two barley plants provide sufficient material for downstream nitrogen analysis
?. Identify and separate the plant roots based on their type. The split root design here was used to separate the embryonic roots (ER) from the remaining roots (RR). Roots were bundled using nylon wire clips (Figure 1).
?. Return the plants to their hydroponic growth tanks.
?. [DAY 21]
On sampling day, between 11:00 and 13:00 hours, remove plants from their tanks and immerse them into the pre-wash solution for 5 min (Figure 1).
?. Transfer the bundled ER to a 15N labeled solution for 10 min (Figure 1).
?. Transfer the bundled ER and the RR to separate tubes of post-label solution for 2 min to avoid contamination of the unlabeled roots with the 15N label (Figure 1).
?. Transfer the bundled ER and the RR to their own 50 ml tubes for a final 5-sec dip in wash solution (Figure 1).
?. Remove the plants and divide into shoots, embryonic roots and non-embryonic roots. Dry them at 70oC for 72 hours. After drying, homogenize the samples into a powder.
?. Maintain a dry powder by storing samples in a desiccator until the preparation of samples for elemental analysis of nitrogen.
?. For elemental analysis of 15N and 14N isotopes, weigh 1-5 mg of sample in a tin capsule and fold.
?. Perform elemental analysis of total nitrogen content using an elemental analyser paired to a mass spectrometer.