Improving the extraction and purification of leaf and phloem sugars for oxygen isotope analyses

The oxygen isotopic composition (here shown as the δ¹⁸ O value) of soluble sugars in leaves and phloem tissue holds valuable information about plant functions in response to climatic changes. However, δ¹⁸ O analysis of sugars is prone to error, and thoroughly tested methods are lacking.

We performed three experiments to test if sample preparation modifies the δ¹⁸ O values of sugars. In experiment 1, we tested the effects of oven-drying versus freeze-drying, whereas in experiment 2 we focused on the extraction and purification of leaf sugars. In experiment 3, we investigated the exudation and purification of twig phloem sugars as a function of exudation time and different ethylenediaminetetraacetic acid (EDTA) exudation media.

Freeze-drying produced more consistent δ¹⁸ O values than oven-drying for sucrose but not for phloem sugars. The extraction and purification of leaf sugars can be performed without a significant modification of their δ¹⁸ O values; yet the purified leaf and phloem sugars possessed higher δ¹⁸ O values than the fraction of water-soluble compounds. Moreover, the exudation time significantly modulated the δ¹⁸ O values of phloem sugars, which is probably related to changes in the sugar composition. The addition of EDTA did not improve the determination of the δ¹⁸ O values of phloem sugars.

We show that the sample preparation of plant sugars for the reliable determination of δ¹⁸ O values requires a strict protocol, which is described in this paper. For phloem sugar, we recommend a maximum exudation time of 1 h to reduce the degradation of sucrose and minimise oxygen isotope exchange reactions between the resulting hexoses and water.