Design and Operation of a Continuous ¹³C and ¹⁵N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

The overall goal of this procedure is to produce plant material that is highly enriched in carbon 13 and nitrogen 15 uniformly throughout the plant or differentially in structural and metabolic tissues. This is accomplished by first constructing an airtight chamber for plant growth. The second step is to control temperature, humidity, and carbon dioxide levels in the chamber to maintain suitable growing conditions for the plants.

Next, the plants are grown in the sealed chamber with air containing 13 C, carbon dioxide and irrigation and fertilization containing nitrogen 15. The final step is to cease labeling by removing the differentially labeled plants from the chamber weeks prior to harvest, so that their metabolic material becomes less enriched in carbon 13 and nitrogen 15. As compared to the structural plant components after harvesting the plant material, hot water extraction and isotope ratio, mass spectrometry are used to measure overall isotope labeling and differences in labeling strength.

The main advantage of continuous labeling over other methods, such as foliar application or repeated pulse labeling, is that it produces uniformly labeled plant material that can be differentially labeled in its metabolic or structural components. To begin this procedure, construct the labeling chamber as described in the accompanying text protocol by mounting 3.18 millimeter thick transparent acrylic walls and a 6.35 millimeter thick transparent acrylic ceiling on an aluminum frame with a white painted steel floor, the dimensions of the chamber can be tailored to suit specific plant growth needs. The chamber shown here measures 1.2 by 2.4 by 3.6 meters and holds 40 15 liter pots ensure that the chamber is airtight by covering all the seams with adequate silicone sealant.

Mount the door onto machine screws, which can be screwed and unscrewed using removable wing nuts. Seal the door with weather stripping to prevent air leakage in an area directly adjacent to the labeling chamber. Mount the control center to the monitor and adjust the temperature, humidity, and carbon dioxide within the chamber.

Ensure that the electrical wires and gas tubing which pass into the chamber are well sealed with silicone to prevent air leakage. Optionally add lights to the chamber to increase light penetration and control day length To regulate chamber temperature. Install a commercial split type air conditioner with the evaporator coils located inside the chamber and the compressor and condenser coils located outside the chamber to dissipate the heat.

Next, install a small room dehumidifier in the chamber. Drill a hole through the floor adjacent to the dehumidifier, and pass the drainage tube from the dehumidifier through the hole and into a jar primed with water. This creates an airtight seal for the drain and allows for pressure E equilibration regulate humidity in the chamber by connecting a programmable controller such as an omega eye series with a humidity sensor to the dehumidifier fire.

Then install an infrared gas analyzer or ER a with a diaphragm pump that continuously draws air from the chamber through the ER A and back to the chamber. This maintains a closed system while continuously monitoring carbon dioxide concentration in the chamber. Next, connect two pure carbon dioxide gas tanks to the carbon dioxide control system.

One of the tanks should contain 10 atom percent, 13 C carbon dioxide or higher, and the other should contain natural abundance. 13 C carbon dioxide. Set the tank regulators to 20 PSI after the regulators insert solenoid valves in the gas lines to control carbon dioxide injections by the Ergo software.

Control the opening and closing of the solenoid valves by connecting them to the ER rga via a solid state relay program, A low alarm on the ergas software to trigger the opening of the solenoid valves when the carbon dioxide concentration in the chamber drops below a certain value and a deadband to close the valves. Once the carbon dioxide concentration reaches an upper set point. After the solenoid valves attach a metering valve to each of the two gas tank lines and carefully adjust them to the desired carbon 13 enrichment level in the atmosphere.

Here we use a 4.4 atom percent carbon 13 enrichment. Join the outlets together and finally pipe the line into the center of the chamber between fans, which helps to distribute the labeled carbon dioxide evenly throughout the chamber to set up the irrigation system. First, create one drip irrigation ring per pot and feed the irrigation tubing through a small hole made in the chamber wall to the exterior.

Then seal the holes around the irrigation tubing with silicone caulking to prevent air leakage on the exterior of the chamber. Connect the irrigation tubing to peristaltic pump tubing and place a small hose clamp on it to prevent air leakage between waterings germinate prior to planting in pots to ensure viability inoculate the seedlings with fresh soil slurry. To introduce beneficial microbes, fill pots with a soil free potting mix to eliminate the introduction of unlabeled carbon and nitrogen from the soil.

Here we use a mixture of sand vermiculite and a profile porous ceramic. Once the seeds have germinated, carefully transplant seedlings to the pots with minimal potting soil. Then move the pots into the chamber and assemble each pot with an individual irrigation hose.

Finally, seal the door to the chamber and scrub the external carbon dioxide by connecting a soda lime scrubber to the air pump until the carbon dioxide concentration is down to at least 200 to 250 parts per million. Before filling the chamber back up to 400 parts per million. Using the 13 C carbon dioxide tank mixture, try to keep the chamber closed through the duration of the growing season to minimize natural abundance carbon dioxide contamination.

First label a hyland's type fertilizer solution with nitrogen 15 by mixing 98 atom percent nitrogen, 15 potassium nitrate with natural abundance nitrogen, 15 potassium nitrate, and then adding it to the rest of the hookin solution. Here we use a seven atom percent nitrogen 15 solution to fertilize. Use a peristaltic pump to feed the nitrogen 15 labeled fertilizer solution through the irrigation tubes.

UNC clamp the irrigation hoses and dispense varying amounts of fertilizer to the individual plants according to demand and experimental design. Then pump water through the hoses to rinse the fertilizer from the irrigation hoses. The total amount of fluid added should not exceed the water holding capacity of the pots in order to minimize fertilizer waste.

Rec clamp, all hoses after fertilization and irrigation to eliminate chamber air leakage. For differential labeling of structural and metabolic components, remove plants from the labeling chamber one to three weeks prior to harvest. Retain plants that are to be uniformly labeled inside the chamber until harvest.

To induce senescence. Simply cease irrigation when ready. Harvest plants by first clipping the above ground biomass.

After harvesting the above ground biomass, pour out the potting mix and roots over a six millimeter core screen to separate out the roots from the potting mix. Then rinse the roots over a two millimeter screen To remove any remaining potting material. Use tweezers as necessary to remove any potting mix that may have clum to the roots, and then allow the roots to air dry in preparation for future experiments.

During three growing seasons of operation, the chamber has successfully maintained the temperature between 26 and 29 degrees Celsius humidity between 36 and 56%and carbon dioxide concentrations between 360 and 400 parts per million as they were set up in the control center. Shown here are representative results from the 2011 growing season of Andra Pogon Girard di. The carbon dioxide buildup during nighttime respiration does not appear to damage the growing plants and is quickly taken up after sunrise.

The nitrogen 15 labeling through the targeted seven adam percent nitrogen 15 hoglin solution produced highly labeled plant material at 6.7 atom percent nitrogen 15, a slight dilution from the targeted nitrogen 15 label may be caused by some natural abundance nitrogen in the potting mix or from the native soil inoculation. The carbon 13 labeling using a targeted 4.4 atom percent 13 C carbon dioxide resulted in 4.46 atom percent, carbon 13. Throughout the whole litter of the uniformly labeled plant material, the differentially labeled plants which were removed from the chamber seven, 14, or 22 days prior to harvest, had a significant difference in carbon 13 and nitrogen.15.

Content in metabolic components obtained as hot water extracts and structural components obtained as hot water extract residues indicating differential labeling of metabolic and structural plant materials. After its development, continuous dual isotope labeling has paved the way for researchers in biogeochemistry to explore the fate and transformation of plant components in the environment at the macro and nano scale.