TITLE: Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues
AUTHORS: Lynn    Doran, Amanda  P. De Souza

[DESCRIPTION]
Ethanolic extraction of total soluble sugars (liquid fraction) and starch (precipitated fraction) from leaf tissue.



[BEFORE_START]
Freeze-dry and grind leaf tissue into a fine powder.
Freeze-dry according to equipment manufacturer's instructions.
Freeze-dried leaf tissue can be ground using the tissuelyzer WITHOUT THE USE OF LIQUID NITROGEN.
Improperly freeze-dried tissue will experience total sugar degradation and significant starch degradation.  Improperly freeze-dried leaf tissue will look oxidized and more brown.

[GUIDELINES]
References: 
Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S, Lee YC. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem. 2005 Apr 1;339(1):69-72. doi: 10.1016/j.ab.2004.12.001. PMID: 15766712. https://pubmed.ncbi.nlm.nih.gov/15766712/

Pak S. Chow, Simon M. Landhäusser, A method for routine measurements of total sugar and starch content in woody plant tissues, Tree Physiology, Volume 24, Issue 10, October 2004, Pages 1129–1136, https://doi.org/10.1093/treephys/24.10.1129

[STEPS]
SECTION: Ethanolic Extraction
1. Weigh 10-15 mg of freeze-dried pulverized material into a labeled, 2 mL screw-cap tube.  Record the weight.
 

SECTION: Ethanolic Extraction
2. Add 1 mL of 80% ethanol to each sample.  A repeat pipettor is useful for large sample numbers.

SECTION: Ethanolic Extraction
3. Vortex to mix the samples until all powder dissolved.
SECTION: Ethanolic Extraction
4. Incubate the samples at 80 °C in water bath for 20 min.

 

SECTION: Ethanolic Extraction
5. Centrifuge the tubes at max speed (>15,000 g) for 3 min to precipitate solids.

SECTION: Ethanolic Extraction
6. Decant the supernatant into  alabeled 8 mL tube.
 

SECTION: Ethanolic Extraction
7. Repeat steps 2-6 four to six times depending on the tissue/plant.  All the supernatants from the same sample should be combined into the correctly labeled 8 mL tube after each centrifugation.

 

SECTION: Ethanolic Extraction
8. Store 8 mL supernatant tubes on ice or at 4 °C while tissue is incubating at 80 °C.

 

SECTION: Starch Precipitate
18. After removing the last ethanolic supernatant, leave the lids off and dry the remaining pellet at 40 °Covernight

 


SECTION: Starch Precipitate
19. After the pellet is dry, replace the lids.  The dried starch pellet can be stored at room temperature.
SECTION: Total Sugar Resuspension
9. After combining the last ethanolic supernatant, remove the ethanol using a Speed Vac Concencentrator, following manufacturers recommendations.
 

SECTION: Total Sugar Resuspension
10. Dry samples on Speed Vac Concentrator just until dry and all traces of ethanol have evaporated.
 

 

SECTION: Total Sugar Resuspension
11. Add 1 mL of distilled or MilliQ water to the dried sugars in the tube.
SECTION: Total Sugar Resuspension
12. If using polystyrene 8 mL tubes, transfer the aqueous sugar solution to a polypropylene, polyethylene, or other organic solvent resistant tube.
 

SECTION: Total Sugar Resuspension
12.1. Vortex the tube for 30 seconds.  Use a pasteur pipette to pipette the liquid vigourously ensuring all sugars in the tube have dissolved in the water.
SECTION: Total Sugar Resuspension
12.2. Leave the tubes in an ice bath on a plate shaker for 60 min to help dissolve all the sugar.

SECTION: Total Sugar Resuspension
12.3. Transfer the entire aqueous sugar solution to a new labeled chemical resistant tube such as a 2 mL polypropylene or polyethylene microcentrifuge tube.
SECTION: Total Sugar Resuspension
13. Add 500uL of chloroform.

 

SECTION: Total Sugar Resuspension
14. Vortex.
SECTION: Total Sugar Resuspension
15. Centrifuge the tubes at max speed (>15,000 g) for 1 min to separate the solvent phases.
SECTION: Total Sugar Resuspension
16. Transfer only the aqueous phase (upper phase) to a new labeled 2 mL tube.
SECTION: Total Sugar Resuspension
17. Store at -20 °C up to several months.