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

Abstract

Rationale The oxygen isotopic composition (here shown as the δ18O value) of soluble sugars in leaves and phloem tissue holds valuable information about plant functions in response to climatic changes. However, δ18O analysis of sugars is prone to error, and thoroughly tested methods are lacking. Methods We performed three experiments to test if sample preparation modifies the δ18O 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. Results Freeze‐drying produced more consistent δ18O 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 δ18O values; yet the purified leaf and phloem sugars possessed higher δ18O values than the fraction of water‐soluble compounds. Moreover, the exudation time significantly modulated the δ18O 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 δ18O values of phloem sugars. Conclusions We show that the sample preparation of plant sugars for the reliable determination of δ18O 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.

Species variation in the hydrogen isotope composition of leaf cellulose is mostly driven by isotopic variation in leaf sucrose

“…To acetylate the extracted plant sugar, 15–30 mg of ground leaf material (that had been microwaved at harvest), was weighed into 2 ml tubes and the water‐soluble compounds extracted in 1.5 ml Milli‐Q water in a water bath set to 85°C (Lehmann et al, 2020). The tube was vortexed and centrifuged and the supernatant transferred to 20 ml glass vials using a syringe with a fitted polyethersulfone filter.…”

A high‐temperature water vapor equilibration method to determine non‐exchangeable hydrogen isotope ratios of sugar, starch and cellulose

“…Cellulose (hemicellulose) was extracted from 100 mg of the fragmented leaf material in F57 fibre filter bags (made up of polyester and polyethylene with an effective pore size of 25 μm; ANKOM Technology, Macedon NY, U.S.A.). In brief, the samples were washed twice in a 5% sodium hydroxide solution at 60 C, rinsed with deionized water, washed 3 times for 10 hr in a 7% sodium chlorite solution, which was adjusted with 96% acetic acid to a pH between 4 and 5, and subsequently rinsed with boiling hot deionized water, and dried overnight in a drying oven at 60 C. The neutral sugar fraction ('sugar', a mixture of sugars, typically glucose, fructose, sucrose and sugar alcohol [Rinne, Saurer, Streit, & Siegwolf, 2012]) were extracted from 100 mg leaf powder and further purified using ion-exchange cartridges, following established protocols for carbon and oxygen isotope analyses (Lehmann et al, 2020;Rinne et al, 2012). This is needed to separate the sugar from other water-soluble compounds such as amino acids which would alter the resulting δ 2 H ne values (Schmidt, Werner, & Eisenreich, 2003).…”

“…The isotopic composition of carbohydrates, which are the primary building blocks of plant biomass, is well known as a useful proxy for hydro‐climatic conditions and plant physiological processes that have occurred during their biosynthesis (Gaglioti et al, 2017; Gessler et al, 2014; Manrique‐Alba et al, 2020; McCarroll & Loader, 2004; Porter et al, 2014; Sass‐Klaassen et al, 2005; Saurer et al, 2012; Saurer, Borella, & Leuenberger, 1997). Various high‐throughput methods have been developed to study the carbon and oxygen isotopic composition of non‐structural plant carbohydrates (NSC; i.e., sugar and starch) (Lehmann et al, 2020; Richter et al, 2009; Wanek, Heintel, & Richter, 2001), and of structural carbohydrates such as tree‐ring or leaf cellulose (Boettger et al, 2007). In contrast, methods to investigate the non‐exchangeable hydrogen isotopic composition (δ 2 H ne ) in plant carbohydrates are still mainly limited to cellulose (An et al, 2014; Arosio, Ziehmer, Nicolussi, Schlüchter, & Leuenberger, 2020; Epstein, Yapp, & Hall, 1976; Filot, Leuenberger, Pazdur, & Boettger, 2006; Mischel, Esper, Keppler, Greule, & Werner, 2015; Nakatsuka et al, 2020; Sauer, Schimmelmann, Sessions, & Topalov, 2009; Xia et al, 2020).…”

High resilience of carbon transport in long‐term drought‐stressed mature Norway spruce trees within 2 weeks after drought release

“…The dried material was milled to fine powder using a steel ball‐mill (Retsch) and about 70 mg per sample were transferred into a 2 ml reaction vial and mixed with 1.5 ml deionized water. The fractions of water‐soluble compounds were then extracted in a water bath at 85°C for 30 min and further purified to neutral sugars using commercial available ion‐exchange cartridges (OnGuard II H, A, & P; Dionex) as described in detail by Lehmann et al (2020). An aliquot of 1 mg of the neutral sugar fraction was then transferred to 5 × 9 mm silver capsules (Saentis Analytical AG), frozen at −20°C, freeze‐dried, and the capsules were closed before isotopic analysis.…”