Protocol

Authors

Petronia Carillo and Yves Gibon

Author Affiliations

Petronia Carillo – Department of Life Science, II University of Naples, via Vivaldi 43, 81100, Caserta, Italy

Yves Gibon – INRA, UMR 619, Biologie du Fruit, 33883 Villenave d’Ornon, France

Overview

This protocol describes how to assay the osmolyte glycine betaine by a HPLC method.

Background

Plants have evolved various developmental, structural or metabolic mechanisms to adapt to salinity. An ubiquitous mechanism involves salt sequestration into the vacuole and accumulation of compatible solutes within the cytoplasm. Glycine betaine (GB) is a well known compatible solute, which has been reported to increase in response to abiotic stresses in many species. GB is an amphoteric compound that is electrically neutral over a wide range of physiological pH values and is extremely soluble in water despite a non-polar hydrocarbon moiety that consists of three methyl groups. In addition to its role as an osmoregulator, GB is thought to interact with both hydrophilic and hydrophobic domains of macromolecules, such as protein and membrane complexes, thus stabilizing their structures and activities against the damaging effects of excessive salt, cold, heat and freezing (Carillo et al. 2008, 2011). Here we describe a method suitable for the determination of GB content by HPLC. This measurement is quantitative and provides reliable data about GB content. The sensitivity of this assay is about 0.05 mM and the linearity is in a range of 0.05-4 mM (i.e. 4-320 nmoles). There is nothing dangerous in this method, but gloves are recommended. Appropriate gloves and face shield or safety glasses should also be worn when handling or dispensing liquid nitrogen.

Materials/Equipment

• Liquid nitrogen
• Safety glasses with side shields and approved cryogenic gloves
• Glycine betaine
• MilliQ grade water
• AG1 8X resin, 200-400 mesh, OH^– form
• Sodium heptane sulfonate
• Na₂SO₄
• H₂SO₄
• 50-100 mL beaker
• 2 or 5 mL plastic syringe
• 8-10 ml polystyrene round bottom test tube
• Centrifuge
• Microtube centrifuge
• 1.5 ml tubes
• HPLC with diode-array spectrophotometer
• ODS2 C18 column (250 mm x 4.6 mm internal diameter)

Units, terms, definitions

Glycine betaine content – μmol g^-1 FW

Procedure

Plant material and extraction

For the following step approved cryogenic gloves and safety glasses are highly recommended

Plant material is frozen in liquid nitrogen immediately after harvesting. Mortar and pestle are pre-chilled in liquid nitrogen and frozen samples are placed in the mortar and grinded gently to a fine powder. Then, the powder is transferred/weighted to several precooled 1.5 mL tubes (eppis) and stored at -80 ^∘C.

The samples (40-50 mg FW) are suspended in 1 ml of MilliQ grade water, subjected to a freeze-thaw cycle by freezing in liquid nitrogen and thawing at 40^∘C for 20 min, and left overnight a 4^∘C. Samples are then centrifuged at 14000 g, 4^∘C for 5 min, and the clear supernatants separated from the pellets. The extraction procedure can be repeated on the pellet and supernatants pooled and used for the analyses. The first extraction, however, allows a recovery over 95%.

If the concentration of GB is too low in the extract, it is possible to dry the clear supernatants (0.8-1 mL) using a vacuum concentrator system and resuspend the samples in a minimal volume of MilliQ grade water (100-200 L). Check the effectiveness of resuspension by vortexing or pipetting up and down until the suspension is clear!

Fractionation of crude extract by ion exchange chromatography

NB: all steps are carried out at 4 ^∘C (Bessieres et al., 1999).

For the following steps gloves are recommended!

In a 50-100 mL beaker the desired amount of cationic exchange resin (AG1 8X, 200-400 mesh, OH^– form) is added and mixed with a similar amount of bidistilled water. The resin slurry is stirred to obtain a homogeneous suspension.

In the bottom of a 2 or 5 mL plastic syringe (or syringe-type column) is placed a porous filter disc (or a cheaper and mono-use disc of filter paper) on which to pack the resin bed.

The plastic syringe is disposed in the polystyrene tube.

In the plastic syringe is placed a sufficient volume of resin slurry to obtain the desired settled resin-bed volume (1.5-2 mL), considering that for each column 3 mL of 50% slurry will result in a 1.5 mL settled (i.e., packed) bed and the resin dried down by centrifugation (3 min, 4 ^∘C, 300 g).

From the bottom of the polystyrene tube the water drained from the syringe is removed.

An aliquot of the soluble extract (125 L) is loaded onto the column, followed by 875 L of MilliQ water.

The eluate is collected by centrifugation (300g at 4^∘C for 3 min) and either analyzed immediately or stored at -20^∘C.

This procedure allows the complete retention of all proline, which is completely absorbed by the anionic resin, and the retention of 80% of other amino acids. It is important to avoid the presence of proline because it has the same retention time of GB (about 4 min) in the HPLC system/run and can interfere with GB quantification. The absence of interfering signals close to that of the GB peak allows a better measurement.

Resin regeneration

For cleaning and recycling the resin, the column is unpacked and the resin washed in batch mode. The used resin (1.6-2 ml) is directly placed into a beaker and stirred in two volumes of 1 M NaOH for 15 min. The NaOH is discarded and the resin washed with 1 M NaCl for 10 min. Then, the resin is washed with distilled water until the pH of the effluent reaches neutrality. After one last washing step with bidistilled water, the resin can be used immediately or stored in bidistilled water at 4^∘C.

Glycine betaine determination

Extract (store at -20^∘C)

Standard: GB solutions ranging from 0.05 to 4 mM, in MilliQ grade water (store at -20^∘C)

HPLC mobile phase: 13 mM sodium heptane sulfonate (2.63 g / L) and 5 mM Na₂SO₄ (0.71 / L) in MilliQ grade water, adjusted to pH 3.7 with H₂SO₄ (the solvent must be degassed).

GB amount is determined by HPLC injecting the extract (20-100 L) onto a ODS2 C18 column (250 mm x 4.6 mm internal diameter) preceded by a pre-column (10 x 1 mm) packed with the same phase. The HPLC mobile phase is delivered by an isocratic pump at a flow rate of 0.8 mL min^-1. The complete run lasts 15-20 min. The eluted GB (retention time 4-5 min) is detected by measuring the absorbance at 200 nm using a diode-array spectrophotometer and quantified by a comparison of peak surface areas with those obtained with pure GB standard solutions in the range 0.05-4 mM.

Calculations:

The absorbance peak area of standard curve will be plotted versus concentration (4-320 nmoles or 0.05-4 mM). If all goes well the curve will be linear (a straight line) (Fig. 1 A, B ). So by measuring the absorbance peak area of extract containing an unknown concentration of GB from plant extract, it is possible to use slope and blank obtained from the calibration curve to estimate the concentration.The HPLC profile of aqueous extract does not show any signal at 4-5 min (Fig. 2 A).

Figure 1. Calibration curves plotted as peak area vs mM GB (A) or as peak area vs nmoles GB (B).

The following equation is used to calculate the amount of GB in the extracts:

GB in nmol mg^-1 FW or in μmol g^-1 FW = Absorbance Peak Area_extract /slope*Vol_extract/Vol_aliquot* concentration factor (if vacuum concentrator is used) *1/FW

Where: Absorbance Peak Area_extract is the peak area (absorbance at 200 nm) determined with the extract, slope (expressed as absorbance nmol^-1) is determined by linear regression, Vol_extract is the total volume of the extract, Vol_aliquot is the volume of extract injected onto the HPLC column, FW (expressed in mg) is the amount of plant material extracted. It is assumed that Abs_extract is within the linear range.

In plants which produce GB, it typically ranges from 0.2-1 (unstressed) to 6-13 (stressed) μmol.g^-1 fresh weight.

Figure 2. HPLC chromatograms obtained from aqueous extract (A) and from young leaf extract of 100 mM NaCl treated durum wheat plants (B).

References

Bessieres MA, Gibon Y, Lefeuvre JC, Larher F (1999) A single-step purification for glycine betaine determination in plant extracts by isocratic HPLC. Journal of Agricultural and Food Chemistry 47: 3718-3722.

Carillo P, Mastrolonardo G, Nacca F, Parisi D, Verlotta A, Fuggi A (2008) Nitrogen metabolism in durum wheat under salinity: accumulation of proline and glycine betaine. Functional Plant Biology 35 (5): 412-426.

Carillo P, Parisi D, Woodrow P, Pontecorvo G, Massaro G, Annunziata MG, Fuggi A, Sulpice R (2011) Salt induced accumulation of glycine betaine is inhibited by high light in Durum wheat. Functional Plant Biology 38 (2): 139-150.

Health, safety & hazardous waste disposal considerations

There is nothing dangerous in this method, but gloves are recommended. Appropriate gloves and face shield or safety glasses should be worn when handling or dispensing liquid nitrogen.