Testing different methods for the extraction and purification of leaf and phloem sugars for oxygen isotope analysis

Oxygen isotope analysis of plant material, such as sugars in different tissues, provides an important tool to understand how plants function, interact with their environment and also cope with climate change. Knowing how to extract and purify carbohydrates without artificially altering their oxygen isotope ratio (δ18O) is therefore essential.We aimed to resolve the impact of different steps on sugars' δ18O values during their extraction and purification from leaf and phloem tissue. More precisely, we investigated (1) different drying processes (oven- vs freeze-drying), and (2) how extraction and purification affect leaf sugars. To clearly see fractionation and exchange processes, these experiments were performed using 18O-labelled water. We further examined (3) the influence of different EDTA media and immersion times to facilitate sugar exudation and subsequent yield from twig phloem tissue. Finally, we analysed (4) the sugar phloem composition, as well as the individual compounds’ carbon isotopic signatures (δ13C).Comparison of freeze- and oven-dried sugars showed lower δ18O memory effects and more consistent oxygen isotopic signatures across different sugars, indicating lyophilisation as the more reliable method. The extraction and purification can be conducted without significant oxygen isotope fractionation. However, 18O-depletion was observed when sugars were dissolved and dried multiple times. This suggests that additional dissolution and drying steps should best be avoided whenever possible. Different immersion times and exudation media during twig phloem extraction revealed to have a substantial influence on the phloem sugars' overall oxygen isotopic signature, their composition, and the individual compounds' δ13C values.Our research illustrates which precautions during sample preparation – from drying to extracting and purifying – need to be taken when plant sugars and their oxygen isotopic signature are of interest. Regarding the preservation of the phloem sugars' original δ18O values and stabilising their composition (prevention of sucrose degradation) as much as possible, we recommend a short immersion time of approx. 1 hour. After a thorough initial rinse of the tissue, the sap should be eluted in pure water without any additives (no EDTA). This further reduces the possibility of hexoses to exchange oxygen with that of the surrounding water.