Jul 13, 2020
Root nitrate influx
- 1[The University of Melbourne];
- 2[The University of Melbourne], [King Abdullah University for Science and Technology]
Protocol Citation: Prajakta Bendre, Vanessa Melino 2020. Root nitrate influx . protocols.io https://dx.doi.org/10.17504/protocols.io.biibkcan
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 13, 2020
Last Modified: July 13, 2020
Protocol Integer ID: 39203
Keywords: Split-root design, root nitrate influx., 15N label,
Abstract
Nitrogen (N) in the form of nitrate (NO3-) 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 NO3- influx with the use of 15N labelled NO3- at a concentration that stimulates the inducible high-affinity nitrate transporters. Stable isotopes such as 15N can be used to trace the movement of NO3- through the plant. This protocol uses a split-root design where only embryonic roots are treated with the 15N 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.
Guidelines
0.1 mM N in the form of K15NO3 was applied to the plants in this protocol. If using potassium nitrate, K15NO3 (Sigma-aldrich), as the label, balance the K+ across all solutions using K2SO4. Each of the pre-label, label and wash steps are performed in 50 ml centrifuge tubes. A single, thin aquarium tube hose is placed in each falcon tube and connected to a pump to provide aeration.
Materials
1. Pre-wash solution:
Use nutrient growth solution (ie. modified strength Johnson nutrient solution, Johnson et al. 1957) maintaining the same treatments supplied during the growth of the plants.
2. Label solution:
Pre-wash solution except that the nitrogen source is replaced with 0.1 mM N in the form of K15NO3. The concentration of all other nutrient elements in the original growth solution are maintained.
3. Post-label solution:
As per pre-wash solution.
4. Wash solution:
0.01 M calcium sulphate.
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
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
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.