Protocol

 

Authors

Hendrik Poorter & Yvonne de Jong-Van Berkel

Author Affiliations

Hendrik Poorter IBG-2, Forschungszentrum Jülich

Yvonne de Jong-Van Berkel, Ecophysiology of Plants, Faculty of Science, Utrecht University

Overview

Soluble sugars are easily extracted from freeze-dried or fresh plant material using 80% ethanol. In oven-dried material, caramelization will lead to an under estimation of soluble sugars. The ethanol extracts can be used immediately for the determination using anthrone reagents. Following the extraction of soluble sugars using 80% ethanol and water, the residue can then be used for the extraction of insoluble sugars such as starch. Starch is extracted using a boiling acid solution of 3% hydrochloric acid. In this way the starch is hydrolyzed to glucose. If you suspect your plants to accumulate fructans, ask yourself which values you want to obtain: all insoluble sugars or starch only. In the latter case use a fructan extraction first. Glucose in the hydrolized extract can be determined colourimetrically using anthrone reagents.

Extraction – soluble sugars

Materials/Equipment

Reagents

• 80% Ethanol
• Chloroform

Equipment

• Water bath
• Table centrifuge

Procedure

1. 50 to 100 mg of dry, ground plant material is extracted with 5 ml 80% ethanol in a centrifugation tube.
2. Or 500 mg of fresh plant material is extracted with 4 ml 80% ethanol in a centrifugation tube.
3. Put the tubes in a water bath for 30 minutes at 30^∘C.
4. Remove the supernatant after centrifugation at 4500 rpm (2649 g) for 10 minutes.
5. Extract the pellet again with 2.5 ml 80 % ethanol, put the tubes in a water bath for 30 minutes at 30^∘C.
6. After centrifugation the supernatant is added to the other supernatant.
7. This ethanol solution contains the soluble sugars: glucose, fructose and sucrose.
8. To remove other compounds, such as chlorophyll and lipids, that can disturb the colour reaction, extract the solution with 5 ml chloroform and 2.5 ml deionized water.
9. Remove the chloroform by centrifugation for 10 minutes at 4500 rpm (2649 g).
10. The ethanol-water fraction is ready to be used for the determination.
11. The residue can be used for extraction of insoluble sugars (fructans and starch).

Notes and troubleshooting tips

1. Extracts of leaf or stem material can have a green colour which interferes with the measurement of the absorption. To remove the green colour, add a bit of magnesium-carbonate and calcium hydroxide (1:2). Mix thoroughly and centrifuge at high speed in a table centrifuge (4500 rpm – 2649 g). Use the colourless supernatant for the determination.
2. When the extract is not used immediately, it is better to evaporate the ethanol and dissolve the residue in a known amount of deionized water.
3. Store in freezer at -20^∘C.

Extraction – insoluble sugars

Materials/Equipment

Reagents

• Hydrochloric acid 3%
• Deionized water

Equipment

• Table centrifuge
• Heating block
• Digestion tubes

Procedure

1. Add 5 ml 3% hydrochloric acid to the test tubes with the residue that was left over after the soluble sugars extraction and mix thoroughly.
2. Add the suspension to 15 ml 3% hydrochloric acid in a test tube of 75 ml (used for digestion methods).
3. Add some boiling stones.
4. Put the tubes for 3 hours in a heating block for digestion tubes at a temperature of 125^∘C.
5. Decant the suspension, after it has cooled down to room temperature, quantitative in a volumetric flask of 50 ml with deionized water.
6. Centrifuge the suspension at high speed and use the supernatant for the determination.

Notes and troubleshooting tips

1. Be aware that this method can also break down a part of the cell wall sugars. Your estimate will likely be an overestimation of the amount of starch.
2. Alternatively, use an enzymatic method to break down starch. If the enzyme mix does not work optimally with your plant material, you will likely have an underestimation of your starch content.

Chemical Determination – soluble and insoluble sugars using Anthrone

Anthrone in sulphuric acid gives a colour reaction with soluble sugars at high temperatures. This colour has an absorption peak at 625 nm. The active form of the reagents is anthranol (the enoltautomere of anthrone) which condensates with a sugar-furfural derivate. The fresh reagent has a yellow colour. In concentrated sugar solutions the colour will become blue, whereas in less concentrated solutions it will be green. The intensity of the reaction is not the same for all soluble sugars. The intensity of the colour of the glucose and fructose condensate is identical. Galactose produces only 54% of the colour of glucose. Pentoses are less sensitive and give an absorption peak at 600 nm. Aliphatic aldehydes can give a red colour. For the determination of the insoluble sugar content the same procedure is followed as with the soluble sugar content.

Materials/Equipment

• Deionized water
• Anthrone
• 96 % Ethanol
• 98 % Sulphuric acid
• Glucose or fructose standard
• Spectrophotometer

Procedure

Preparation of reagents

1. Dissolve 0.2 g of anthrone in 8.3 ml 96% ethanol and 20 ml deionized water
2. Add carefully 100 ml of 98% sulphuric acid while the solution is constantly cooled on ice
3. Mix the solution until the anthrone is dissolved: keep the solution cooled
4. Store the reagents in a dark bottle in the fridge
5. The reagents must be yellow

Standards

1. Dissolve 11.0 mg d-glucose, d-fructose or sucrose in 100 ml deionized water
2. Dilute in a range from 0, 0.011, 0.022, 0.044, 0.066, 0.088 and 0.110 mg/ml

Determination

1. Cool the test tubes on ice, so the condensate does not disintegrate
2. Add 0.2 or 0.5 ml extract or standard to a test tube
3. Add 5 ml anthrone reagent to the sample or standard
4. Mix well and put the test tubes in boiling water for 7.5 minutes to determine total soluble sugars
5. Then immediately cool on ice
6. Allow the test tubes to warm up at room temperature
7. When cooled, the absorbance is measured at 625 nm .
8. The content of fructose can be determined, using the same procedure but placing test tubes for 15.0 minutes at a water bath of 40^∘C instead of 7.5 minutes in boiling water.

Notes and troubleshooting tips

• Prepare a fresh anthrone solution every 2 days. Exposed to light and higher temperature, the reagent disintegrates rapidly.

Reference values Total Non-structural Carbohydrates (TNC; soluble plus insoluble sugars)in leaves of herbaceous species: 100-250 mg g^-1. Values in stems and roots can be higher. Values for woody species are generally somewhat lower than for herbs. TNC concentration in seeds and fruits can exceed 800 mg g^-1 (Poorter & Villar 1997).

Literature references

Curtis, P.S., Zhong, H.L., Läiuchli, A. and Pearcy, R.W., 1988. Carbohydrate availability, respiration and the growth of kenaf (Hibiscus cannabinus) under moderate salt stress. Amer. J. Bot. 75:1293-1297.

Fales, F.W., 1951. The assimilation and degradation of carbohydrates bij yeast cells. J. BioI. Chern. 193: 113-124.

Gifford, R.M. and Thorne, J.H., 1985. Sucrose concentration at the apoplastic interface between seed coat and cotyledons of developing soybean seeds. Plant Phys. 77:863-868.

Gottschalk, A., 1941. Aust. J. Exp. BioI. Med. Sc. 19: 211.

Hassid, W.Z., Joslyn, M.A. & McCready, A.M., 1941. J. Am. Chem. Soc. 63: 295.

Morris, D.L., 1948. Quantitative determination of carbohydrates with dreywoods anthrone reagent. Science 107: 254.

Hewitt, B.R., 1958. Spectrophotometric determination of total carbohydrate. Nature 182: 246- 247.

Poorter, H & Villar, R. 1997. The fate of acquired carbon in plants: chemical composition and construction costs. In: Plant Resource Allocation. Academic Press, pp 30-72.

Wolswinkel, P., Kraus, E. and Ammerlaan, A., 1986. Effect of the osmotic environment on the balance between uptake and release of sucrose and amino acids by the seed coat and cotyledons of developing seeds of Pisum sativum. J. Exp. Bot. 37: 1462-1471.

Yemm, E.W. & Willis, A.J., 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem. J. 57: 508-14.