Separation of sugars, polyols, proline analogues, and betaines in stressed plant extracts by high performance liquid chromatography and quantification by ultra violet detection

Abstract

Osmoprotectants such as (i) sugars and sugar alcohols (polyols), (ii) proline and its analogues, and (iii) a number of quaternary ammonium compounds (betaines) play a significant role in plant adaptation to environmental stresses. Lack of a simple and rapid technique for the extraction and simultaneous determination of these osmoprotectant solutes from a large number of samples originating from plant breeding populations and agronomic trials, led to the development of this method.

Osmoprotectants were extracted using methanol-chloroform-water. Extracts were partially purified, where required, by treating with ion exchange resins. Solutes were separated using a high performance liquid chromatograph fitted with a Sugarpak-1 column and 5 mg L^-1 Ca-EDTA solution as the mobile phase. The eluted solutes were quantified by ultra violet detection at 195 nm. This technique measures sugars/polyols, proline analogues, and betaines in a variety of plant species such as peanut, Melaleuca uncinata, and cotton, respectively. Pinitol levels in peanut estimated using this method correlated significantly with the determinations obtained by HPLC-refractive index determination (R² = 0.983, P<0.001). Glycinebetaine determinations in cotton using this method were significantly correlated with determinations achieved by ¹H NMR spectroscopy (R² = 0.989, P<0.001). Cotton leaf extracts spiked with a range of authentic glycinebetaine levels were also precisely measured using this technique (R² =0.999, P<0.001). The described method is simple, rapid, sensitive, cost effective and simultaneously measures more than one class of osmoprotectants from a single chromatographic run.