Rationale

The anthrone method has been extensively used for the colorimetric quantification of carbohydrates. The most common sugars are glucose and fructose (isomer monosaccharides) and the disaccharide sucrose, formed by a molecule of glucose and a molecule of fructose. Glucose is produced by photosynthesis in plant cells and is a primary source of energy in the cells of all living organisms whilst sucrose is the form in which sugar is usually transported throughout plants. Glucose, fructose and sucrose are usually known as total soluble sugars (TSS) since they are water soluble. These compounds represent a supply of energy that is immediately available for plant needs. Starches are polysaccharides constituted by many glucose units bonding together and represent the main carbohydrate reserve in plants. The soluble sugars and starches also play an important role in osmoregulation of plants.

This colorimetric assay is based on the reaction of anthrone with the furfural molecules resultant from oxidation of glucose. Disaccharides or polysaccharides (e.g. starches) are first hydrolysed under acidic conditions to form glucose.

Preparation

• [ ] Preserve the samples at –80°C or liquid nitrogen. Keep them away from light.
• [ ] Measure the weight of tissue sample (~50 mg fresh weight, FW).
• [ ] Lyophilise the sample in an envelope covered with aluminium foil for minimum 2 days.
• [ ] Measure the weight of tissue sample (dry weight, DW).
• [ ] Measure the area of the leaf.
• [ ] Transfer the sample into a 15 ml Falcon tube. Store at **–**20°C.

Reagents for TSS determination

<aside> 💡 Always prepare enough reagents for extra 10 samples.

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• [ ] For each sample, prepare:
  • [ ] 5 ml 80% (v/v) Ethanol, which is made by:
    • [ ] 4 ml Water
    • [ ] 1 ml Absolute ethanol
  • [ ] 1.5 ml Anthrone solution, which is made by (the following proportion makes a 21 ml solution sufficient for 13 samples):
    • [ ] 0.04 g Anthrone powder
    • [ ] 1 ml Water
    • [ ] 20 ml Concentrated sulphuric acid

<aside> ⚠️ Concentrated sulphuric acid is extremely corrosive and dangerous. Handle the reagents with care. Discard the waste carefully by first diluting it in a very large volume of water.

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D-glucose standards

<aside> ⚠️ Standard curves need to be prepared with excellent precision and care to yield reliable results.

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