Colorimetric Assay for the Quantification of Non-Structural Carbohydrates in Plant Tissues

1. Plant Collection and Processing

1. After collecting plant samples, flash-freeze samples by dipping plant material into liquid nitrogen with forceps and store at -80 °C. If the plant samples collected are too large to flash-freeze, quickly cool the samples using dry ice and transfer to a -80 °C freezer as soon as possible. The macronutrient content of plant material can change after tissues are separated from the plant, so it is important to freeze plant samples as soon as possible after collection.
   CAUTION: Liquid nitrogen can cause severe frostbite when in contact with skin. Please make sure to transport liquid nitrogen in approved containers and wear proper PPE during its handling, including cryo-gloves, eye goggles, face shield, apron, and a lab coat.
2. Lyophilize plant material using a freeze-dryer to ensure that the tissues are metabolically inactive while water is being removed. Dry until the mass of the sample stabilizes to ensure all water has been removed.
3. Once samples are dry, grind into a fine powder using a grinder or mill. Store samples in a desiccating cabinet until analysis.

2. Digestible Carbohydrate Assay

1. Sample Preparation
   1. Weigh out replicate samples of each tissue, approximately 20 mg each, into glass 15 mL tubes with rubber-lined screw caps (100 mm length). These samples will subsequently be referred to as unknown samples. Label tubes with a waterproof marker or with labeling tape on the lids and record the exact mass of each sample, as this information will be required to calculate the %carbohydrates in each unknown sample.
2. Extract digestible carbohydrates from each unknown sample.
   1. Add 1 mL of 0.1 M H₂SO₄ to each tube and screw caps onto tubes tightly. Place in a boiling water bath for 1 hour.
      CAUTION: Sulfuric acid is very corrosive. Please wear gloves, eye goggles, and an apron when handling H₂SO₄, and perform this step in a fume hood.
   2. Cool off tubes in a tepid water bath. Pour tube contents into labeled 1.5 mL microcentrifuge tubes (do not be concerned if some plant material remains in the glass tubes).
   3. Centrifuge tubes at 15,000 x g for 10 minutes. Remove the supernatant liquid with a micro-pipettor and place into new labeled 1.5 mL microcentrifuge tubes. Tubes can be refrigerated overnight at this point. If continuing with assay, refer to step 2.3.2.
3. Mix standard solutions and quantify the total digestible carbohydrate content of unknown samples.
   1. Prepare D(+) glucose standard solutions with the concentrations listed in Table 1. Prepare six glass test tubes with 400 µL of each standard solution.
   2. Pipette 15 µL of each unknown sample into its own test tube and add 385 µL of distilled water to each for a total volume of 400 µL in each test tube. In a fume hood, add 400 µL of 5% phenol to each standard and unknown sample test tube, and then quickly pipette 2 mL of concentrated H₂SO₄ into each tube. Do not touch the test tube contents with the pipette tip, just add to the solution surface (a repeater pipette works best for this).
   3. Let tubes incubate for 10 minutes, and then carefully vortex the tubes to mix contents. Incubate for an additional 30 minutes.
      CAUTION: Phenol and sulfuric acid are corrosive irritants. To protect from exposure to skin, eyes, and inhalation, this step should be performed in a chemical fume hood using the proper PPE, which includes: face shield or eye goggles, rubber gloves, lab apron, and boots. Mixing phenol with sulfuric acid also results in an exothermic reaction, which will result in the tubes becoming hot.
   4. Pipette 800 µL of sample from each tube into each of 3 polystyrene 1.5 mL semi-micro cuvettes (3 technical replicates per sample). Set the spectrophotometer to read at 490 nm. Calibrate to a blank cuvette containing distilled water before reading standards or unknown samples, and intermittently throughout sample readings. Run each cuvette through a spectrophotometer and record the absorbance. Average across technical replicates for each unknown sample.
      NOTE: In some cases, samples may surpass the upper absorbance threshold. If so, samples should be diluted at step 1.3.1. Dilutions must also be accounted for in subsequent calculations.
   5. Calculate the average absorbance for each standard.
      NOTE: To calculate the amount of total digestible carbohydrates present in each unknown sample using the standard curve, it is easiest to regress the average absorbance of each standard against the micrograms of D(+)glucose present in each standard, but one can also plot the D(+)glucose concentration (µg/µL) of each standard if desirable. The following calculations, however, refer to a standard curve based on micrograms, not concentrations (µg/µL).
   6. Calculate the standard curve by plotting the average absorbance against the total amount of D(+)glucose (µg) in each standard solution. Fit a linear regression line to these data, and then use the following equation to calculate the total amount of carbohydrates (µg) in each unknown sample:
      M_c = (((A_x – b)/m)/(15))*1000
      M_c = µg of carbohydrates in sample
      A_x = average absorbance of unknown sample
      b = y-intercept (standard regression line)
      m = slope (standard regression line)
      The correlation coefficient of this line should be greater than 0.98 and routinely near 0.99. Then, use the following equation to determine the percentage of carbohydrates in each sample:
      P_c = ((M_c/1000)/(M_i))*100
      P_c =%carbohydrates
      M_i = initial mass of sample (mg)

Table 1. Standard curve calculation for digestible carbohydrate assay. The amount of glucose in each standard is calculated by taking the concentration of each standard and multiplying it by the amount of standard solution in each test tube (400 µL)