Protocol

 

Authors

Damesin C^1,2, Bazot S^1,2, Fresneau C^2,1

Author affiliations

1. Université Paris-Sud, UMR 8079, Laboratoire Ecologie Systématique et Evolution, Orsay, F-91405, Orsay, France

2. CNRS, UMR 8079, Laboratoire Ecologie Systématique et Evolution, Orsay, F-91405, Orsay, France

Overview

The present extractions are easy and quick methods with water for soluble sugars and with HCl/methanol for starch. The protocol is the result of adjustments achieved by different persons from an inherited one.

Background

Extractions require four days for 48 samples* (or three days for 24 samples) without considering the time needed with HPLC (High Performance Liquid Chromatography).

Materials/Equipment

• Centrifuge
• Fridge (around 4^∘C)
• Freezer (-20^∘C or -80^∘C)
• Heater
• Evaporator-concentrator
• Vortex
• Balance
• HPLC
• Pipetman (automatic pipette)
• Plastic centrifuge tube 10 mL (Nalgene)
• Glass centrifuge tube 50 mL (Kimble Chase)
• 1.5 and 2 mL microtubes with and without screw caps
• Biochemistry: fresh water, HCl, methanol, ethanol

Procedure (for 48 samples)

1. Soluble sugar extraction (start in the morning of day 1 for the first 24 samples then start the next 24 samples in the afternoon)

1. Weigh about 50 mg of finely ground plant tissue (record the weight) in 1.5 mL microtubes with screw cap.
2. Add 1 mL fresh H₂O, vortex 30 s and place on ice 10 min.
3. Vortex 30 s and then wait again for 10 min on ice.
4. Centrifuge at 12 000 g, 5^∘C for 10 min.
5. Transfer the supernatant with a Pasteur pipette, into another microtube with screw cap.
6. Place the pellet in a freezer (-20^∘C) for later starch extraction.
7. Place tubes with the supernatant in a heater at 100 ^∘C for 3 min in order to denature all proteins.
8. Transfer tubes 3 min on ice.
9. Centrifuge at 12 000 g, 5^∘C for 10 min to eliminate the denatured proteins (the pellet contains proteins).
10. Collect the supernatant in 1.5 mL microtubes (simple 1.5 mL tubes), this fraction is used for sugar analysis.

2. Soluble sugar quantification (HPLC analysis)

1. Filter the supernatant through 0.45-μm filters (Millex-HV, Nihon Millipore, Kogyo, Japan).
2. Inject 25 L of the filtered extract into a high-pressure liquid chromatography apparatus. Sucrose, glucose and fructose were separated by a Sugar Pak1 column (300 mm x 6.5 mm; Waters, Guyancourt, France) equipped with a carbohydrate pre-column (7.5 mm x 4.6 mm; Alltech, Templemars, France). The flow rate is fixed at a constant 0.5 mL min ¹, which maintains the pressure at around 40 bars and the temperature of the column is 90^∘C. Individual carbohydrates were eluted from 7 to 13 min after injection. Peaks for the three sugars (glucose, fructose and sucrose) are detected using a refractometer (refractive index detector 133; Gilson, Villiers-le-Bel, France). Sugar concentrations are determined as the areas under the curves, compared with a range of standards of the three sugars.
3. The same process was used for preparing isolated, pure fractions of the different sugars for isotope analysis. To obtain enough material, 200 μL of filtered extracts were applied to the column. Each sugar (glucose 7 min, fructose 10 min and sucrose 12 min approximately) is separately harvested in a microtube. Eluats are lyophilised, and dried sugars are solubilised in 50 μL fresh water and transferred in tin caps (6×8 mm) for IRMS analysis.

3. Starch extraction and quantification (three step process)

First step: pellet wash (start in the morning of day 2 for the first 24 samples, then start the next 24 in the afternoon)

1. Place on ice previously frozen tubes with the pellet and add 1 mL of ethanol.
2. Vigorously vortex 30 s (check the complete dissolution of the pellet).
3. Place tubes in the heater at 70^∘C for 10 min.
4. Centrifuge for 10 min at 12 000 g, 5^∘C.
5. Discard the supernatant.
6. Repeat these steps as many times as needed until total loss of any color in the supernatant (about 2-3 times for poorly chlorophyllous tissue like woody tissue, around 10 times for rich chlorophyll tissues like leaves).
7. Place tubes with washed pellets in a freezer (-20 ^∘C)

Second step: solubilisation and flocculation of starch (start in the morning of day three for the first 24 samples, then start the next 24 samples in the afternoon)

1. Place microtubes with washed pellet on ice.
2. Add 1 mL of 6M HCl (100 mL preparation: 53 mL of 35% HCl + 47 mL H₂Od).
3. Vortex 30 s and transfer the mixture into a 10 mL plastic tube.
4. Rinse microtube with 1 mL HCl and transfer again into the 10 mL tube.
5. Leave 1h at 5^∘C (on ice).
6. Centrifuge at 12 000g, 5^∘C for 20 min.
7. Transfer the supernatant with a Pasteur pipette into a 50 mL glass tube.
8. Add 2 mL of HCl on pellets (in10 ml tube), vortex 30 s.
9. Leave 1 h at 5 ^∘C.
10. Centrifuge as above and recover the supernatant as previously.
11. Add 16 mL methanol in glass tubes (1:4 v/v) and vortex.
12. Leave tubes 1 night at 5^∘C (in the fridge).

Third step: recovery and quantification of starch (day 4)

1. The following morning, centrifuge glass tubes at 11 000 g, 5^∘C for 30 min.
2. Immediately after centrifugation, carefully discard the supernatant.
3. Add methanol in several times (until 2 mL) to rinse the tubes and remove all starch.
4. Transfer the mixture each time on a previously weighted 2 mL microtube with a Pasteur pipette.
5. Centrifuge at 12 000 g, 10 min, 5^∘C. Discard the supernatant.
6. Dry at 30^∘C in an evaporator-concentrator (to evaporate methanol) during 1 h and weigh the closed tubes after 15 min to determine the quantity of starch.
7. The pellet is then transferred in a tin cap (4×6 mm) for IRMS analysis.

Other resources

Comparisons of starch and sugar extraction for isotope analysis could be found in Richter et al. (2009)

Notes and troubleshooting tips

• To ensure accuracy of this gravimetric quantification of starch, the chosen tubes should be rather light (e.g. microtubes rather than 10 or 50 mL tubes).
• To avoid problem with humidity, the microtube weights should be carefully done in the same conditions before and after extraction (e.g. microtubes dried at 50^∘C, closed and weighed 15 min after the end of drying).
• The days are busy with 48 samples, to be more relaxed the tissue weighing (see 1.1) can be done beforehand.

Literature references

Damesin C, Lelarge C (2003) Carbon isotope composition of current-year shoots from Fagus sylvatica in relation to growth, respiration and use of reserves. Plant, Cell and Environment 26: 207-219

Duranceau M, Ghashghaie J, Badeck F, Deléens E, Cornic G (1999) Delta ¹³C of CO₂ respired in the dark in relation to delta ¹³C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought. Plant, Cell and Environment 22: 515-523

Richter A et al. (2009) Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods. Rapid Communications in Mass Spectrometry 23: 2476-2488

Health, safety and hazardous waste disposal considerations

Starch extraction has to be conducted under chemical fume hood (pellet wash with ethanol and extraction with HCl and methanol). HCl and methanol wastes should be collected in a clearly labelled plastic closed bottle which can be kept by the security services.