Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Summary

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

Abstract

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Introduction

Plant biomass forms the foundation of virtually all terrestrial food-webs. Plants acquire nutritional elements from the soil through their roots systems and utilize sunlight in their foliar tissues to synthesize biomolecules. In particular, carbon and nitrogen are used to create carbohydrates, proteins (comprised of amino acids), and lipids that are needed to build plant biomass (it should be noted that in plant physiology the term "macronutrient" often refers to soil elements, such as N, P, K, and S, however, throughout this paper this term will refer to biomolecules, such as proteins, carbohydrates, and lipids). When herbivores consume plant material, the macronutrients contained in plant tissues are broken down into their constituent parts and then used to drive the physiological processes of the consumer. In this way, plant macronutrients have a strong influence on consumer physiology along with important implications for higher order ecological interactions and food-web dynamics.

Across the animal kingdom, soluble protein and digestible carbohydrates are the macronutrients most closely tied to survival, reproduction, and performance¹. Moreover, the majority of animals actively regulate their intake of these two macronutrients to meet their physiological demands^1,2. This is particularly true for insect herbivores that detect the concentrations of sugars and amino acids in plant tissues, which in turn directs feeding behavior. As a result, plant soluble protein and digestible carbohydrate content has played a strong role in the evolution of plant-insect interactions.

While data on plant soluble protein and digestible carbohydrate content are relatively rare (but see^{6,7,8,9,10,11}), there is a preponderance of data available on plant elemental content (carbon, nitrogen, and phosphorus). Largely this is because elements play a primary role in plant mineral nutrition^3,4,5. Where elements are measured, correlations have been used to extrapolate the amount of soluble protein and digestible carbohydrate, but accurate calculations are often difficult to obtain. For instance, it is impossible to use carbon as an indicator of plant digestible carbohydrate content because carbon is ubiquitously present in all organic compounds. A stronger relationship exists between elemental nitrogen and plant soluble protein content, and generalized nitrogen-to-protein conversion factors are often utilized. However, there is strong evidence that nitrogen-to-protein conversions are highly species-specific^12,13,14,15, making the use of generalized conversion likely inaccurate. Because of this, nitrogen-to-protein conversion factors often lack precision, particularly to the extent that is required for nutritional studies on herbivores. Also, the presence of N-containing plant allelochemicals, such as alkaloids and glucosinolates that are often toxic to herbivores, can confound these conversions.

Here, we offer two chemical assays for measuring the concentration of soluble proteins and digestible carbohydrates in plant tissues. These assays are presented separately, but it is suggested that they be used concurrently to analyze the same plant samples in order to achieve a more comprehensive analysis of plant macronutrients. Both employ similar methodologies, consisting of an extraction step, followed by quantification via absorbance. Plant sample prep is also identical for both protocols, making it easy to run both analyses in tandem. The utility of these assays do not stem from their novelty, as they rely on older, (Bradford, Jones, Dubois) well-established colorimetric assays^16,17,18, but here we have organized a clear and easy-to-follow protocol that combines these methods with more obscure plant-specific extraction techniques^17,19 in order to make the application of these assays more accessible to those in plant-relevant fields.

For both assays, plant macronutrients are first extracted physically by freezing, lyophilizing, and grinding the plant material. For the soluble protein assay, further chemical extraction is done^17,19 through several rounds of vortexing and heating samples in NaOH solution. The well-known Bradford assay, utilizing Coomassie brilliant blue G-250, is then used to quantify soluble proteins and polypeptides between 3,000-5,000 Daltons^16,17. This assay has a detection range between 1-20 µg total proteins per microplate well or <25 µg/mL, but does not measure free amino acids. The extraction step of the digestible carbohydrate assay is based on the dilute acid method of Smith et al.²⁰ and allows for the isolation of soluble sugars, starch, and fructosan – but not structural carbohydrates. A phenol-sulfuric acid quantification method is taken from Dubois et al.¹⁸ and measures all mono-, oligo-, and polysaccharides (as well as methyl derivatives). This assay is able to quantify specific sugars, but here we use it as an indicator of total digestible carbohydrate content (see Smith et al.²⁰ for more detailed analysis). Together, these assays measure the two macronutrients that are strongly tied to plant eco-physiology and herbivore performance, providing important data on resource quality at the base of terrestrial food-webs. Presenting these protocols promotes the generation of plant macronutrient datasets in order to obtain a more thorough understanding of plant physiology, herbivore nutritional ecology, and plant-herbivore interactions.

Protocol

1. Plant Collection and Processing

1. Collect and process plant samples
   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 googles, 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. Soluble Protein Assay

1. Sample Preparation
   1. Weigh out replicate samples of each tissue, approximately 20 mg each, into 1.5 mL polystyrene microcentrifuge tubes and label tubes. These samples will subsequently be referred to as unknown samples. Record the exact mass of each sample, as this information will be required to calculate the %soluble protein in each unknown sample. Use microcentrifuge tubes with locking lids, as non-locking lids may open during the subsequent heating step, resulting in loss of sample solution.
2. Solubilize and isolate unknown sample proteins
   1. Using a micro-pipettor, add 500 µL of 0.1 M NaOH to each tube. Close lids tightly and sonicate for 30 minutes. Preheat a hot water bath 90 °C.
      CAUTION: Sodium hydroxide is a strong base and can cause burns when in contact with skin. Although 0.1 M NaOH represents a low concentration, wear protective gloves to avoid skin irritation.
   2. Place tubes in a rack in the hot water bath for 15 minutes.
   3. Centrifuge tubes at 15,000 x g for 10 minutes. Pipette the supernatant liquid into new labeled microcentrifuge tubes, using a new pipette tip for each sample. Add 300 µL of 0.1 M NaOH to the tube containing the pellet and centrifuge again at 15,000 x g for 10 minutes. Again, collect the supernatant liquid and combine it with the other supernatant fluid from the same sample. This is a potential stopping point, and samples can be stored at 4 °C overnight if needed.
3. Precipitate plant proteins
   1. Neutralize the pH of the supernatant solution by adding 11 µL of 5.8 M HCl. Confirm a pH of ~7 by dipping litmus paper into each sample solution.
      CAUTION: Hydrochloric acid is a strong corrosive acid that can cause burns when in contact with skin (skin application or inhalation). Please wear gloves to prevent skin irritation while handling HCl.
   2. Add 90 µL of 100% trichloroacetic acid (TCA) to each tube and incubate tubes on ice for 30 minutes. The precipitation of proteins will turn the solution from clear to opaque. If this does not occur after 30 minutes, refrigerate the tubes for 1 hour. This is a potential stopping point, and samples can be stored at 4 °C overnight if needed.
      CAUTION: Trichloroacetic acid is corrosive. Please wear gloves when handling to prevent contact with skin and avoid inhalation.
   3. Centrifuge samples at 13,000 x g for 10 minutes at 4 °C. Carefully remove TCA supernatant by suctioning it out using a vacuum line attached to a fine glass micropipette tip (the same tip can be used across samples). Do not disturb the protein pellet by avoiding heavy suction and keeping an appropriate distance between pipette tip and the pellet.
   4. Wash pellet quickly with 100 µL of -20 °C acetone. This step removes any remaining TCA from the pellet, which can interfere with the subsequent Bradford assay; however, acetone will start to dissolve the protein pellet if left in contact too long, so acetone should be added then quickly extracted from the pellet within 5 seconds.
   5. Allow acetone to evaporate by placing tubes in a fume hood or refrigerator. Then, dissolve the protein pellet by adding 1 mL of 0.1 M NaOH to each tube. Fully dissolving the pellet may require several rounds of heating in a hot water bath, vortexing, and sonicating.
      Note: The longer the tubes sit in the fume hood or refrigerator and the drier the pellets get, the more difficult they will be to re-solubilize. It is suggested to dry the pellet for 30 minutes, and then check for the presence of acetone every 10-15 minutes until it is clear that the acetone has evaporated (no acetone fumes are detectable).
4. Mix standard solutions, dilute unknown sample solutions, and quantify the total protein content of unknown samples using the Bradford assay.
   1. Prepare bovine immunoglobulin G (IgG) standard solutions according to the concentrations listed in Table 1 (lyophilized or IgG stock solution can be mixed with deionized water to achieve each concentration). Store standards at 4 °C. In a 96-well plate, add 160 µL of each IgG standard solution in triplicate starting at the A1 position of the well plate (0 µg/µL in A1, A2, A3 position, 0.0125 µg/µL in B1, B2, B3 positions, etc.).
   2. Prepare a diluted NaOH solution by adding 50 µL of 0.1 M NaOH to 950 µL of distilled water. As a blank negative control, add 60 µL of this solution to the well plate in triplicate (G1, G2, G3 positions).
   3. Prepare dilutions of unknown samples by adding 50 µL of each sample solution to 950 µL of distilled water in a new 1.5 mL microcentrifuge tube. Then, add 60 µL of each diluted sample to the well plate in triplicate, starting at the H1 position. A 96-well plate should allow for the analysis of 6 standards, 1 blank, and 25 unknown samples when read in triplicate. Each well plate analyzed should contain its own IgG standard and blank samples.
   4. Add 100 µL of distilled water to all blank and unknown samples wells. Now each well should contain a total of 160 µL. Using a multi-channel pipette (8 channels), add 40 µL of Coomassie brilliant blue G-250 protein dye to every well in the first column of the well plate. Mix the dye with the samples by plunging several times. Repeat for all other columns, replacing pipette tips to avoid contamination.
      CAUTION: Coomassie brilliant blue G-250 is an irritant and contact with skin, eyes, or lungs should be avoided. Please wear gloves to prevent contact with skin. If contact with skin occurs, this product will stain.
   5. Use a needle to pop any bubbles present in the wells, as they may interfere with the microplate spectrophotometer reading. Clean the needle to avoid contamination between wells. Let the microplate incubate at room temperature for 5 minutes.
   6. Using a microplate spectrophotometer, record the absorbance values for each well at 595 nm.
   7. Record the average absorbance for the three blank wells and subtract this value from each of the standard and unknown sample readings. Then, record the average absorbance for each standard and unknown sample.
      Note: To calculate the amount of protein present in each unknown sample using the standard curve, it is easiest to regress the average absorbance of each standard against the micrograms of protein present in each standard, but one can plot the protein concentration (µg/µL) of each standard if desirable. The following calculations, however, refer to a standard curve based on micrograms, not concentrations (µg/µL).
   8. Plot the average absorbance of each standard against the total amount of protein present in each standard well (Table 1). Fit a linear regression line to these data and use the equation to calculate the amount of protein (µg) in each unknown sample well based on the average absorbance (Figure 1a).
      Note: The correlation coefficient (r value) of this line should be greater than 0.98 and routinely near 0.99. The standard regression will be specific to each plate, hence the unknown sample wells in each plate must be analyzed separately.
   9. Once the average amount of protein is calculated for each unknown sample well, use the following equation to calculate the total amount of protein (µg) in each original sample:
           M_p = (((W_p/60) x 1000)/50)*1000
           M_p = µg of protein in original sample
           W_p = µg of protein in unknown sample well
      1. Then, use the following equation to determine the percentage of protein in each sample:
              P_p = ((M_p/1000)/M_i)*100
              P_p =%protein in sample
              M_i = initial mass of sample (mg)
         Note: In some cases, the sample may surpass the upper absorbance threshold. If so, sample solutions may require further dilution at step 2.4.3.
         Additional dilutions must then be accounted for in subsequent calculations. Some microplate reader brands also provide computer software that will automatically calculate the average protein concentration of each unknown sample based on the standard curve data.

3. 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 sample will subsequently be referred to as unknown samples. Label tubes with a waterproof marker or with labelling 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 googles, 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 3.3.2.
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 2. 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 3.2.3. 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)

Results

To show the usefulness of these methods, we analyzed the soluble protein and digestible carbohydrate content of four different field and sweetcorn tissues that serve as distinct potential nutritional resources for insect herbivores. We collected ears of corn from three agricultural regions in the United States (Minnesota, North Carolina, and Texas), encompassing five different varieties of sweet corn (i.e., genotypes) and one variety of field corn as an outgroup. Table 3<...

Discussion

By combining well-established colorimetric assays with effective plant-specific extraction protocols, the assays demonstrated here provide a reasonable and accurate method for measuring plant soluble protein and digestible carbohydrate content. Our results using corn as an exemplar illustrates how these protocols can be used to obtain precise measurements across different biologically-relevant spatial scales. For example, we were able to detect differences in plant soluble protein and digestible carbohydrate content betw...

Materials

Name	Company	Catalog Number	Comments
microplate reader (spectrophotometer)	Bio-Rad	Model 680 XR
Bio-Rad Protein Assay Dye Reagent concentrate	Bio-Rad	#5000006	450mL