Inter-comparison of extraction methods for plant water isotope analysis and its indicative significance

Abstract

Recent studies have shown that the widely used water extraction method, cryogenic vacuum extraction (CVE), may be biased toward plant water isotope analysis, and the long-standing hypothesis of non-isotopic fractionation between xylem water and sources is being challenged. Thus, there is an urgent need to find alternative approaches for extracting plant water and further verify whether the isotopic composition of water is conserved during its passage from the soil to the plant. To determine the correct methods for extracting plant water for isotope analysis, we performed an inter-method comparison of four plant water extraction techniques on sap-labeled stems and irrigated-pot Malus pumila Mill saplings with known isotopic composition water. Next, we assessed isotope fractionation during root water uptake using an accurate isotope sampling method. Generally, mechanical methods outperformed CVE in recovering the isotopic signals of xylem water. For sap-labeled stems, gas push (GP), centrifugation (CE), and high-pressure mechanical squeezing (HPMS) produced water were not significantly different from the labeling water in 2H and 18O (P > 0.05), while the CVE method induced significant depletion of 2H (Δδ2H = -8.21 ± 0.67 ‰, P < 0.01) and 18O (Δδ18O = -0.79 ± 0.34 ‰, P < 0.05). For irrigated-pot saplings, GP- and CE-obtained water was the closest to the irrigation water isotopically, followed by HPMS and CVE, while the CVE method had the highest extraction success rate (100%), followed by CE (90%), HPMS (50%), and GP (30%). Based on the accurate sampling technique (CE method), we found that 2H and 18O compositions of the labeled-sap water were conserved even when standing in vessels for 24 h. Moreover, CE-obtained xylem water, representing the sap in vessels, showed no significant differences in hydrogen and oxygen isotopic compositions to that of irrigation water. The GP and CE methods can selectively extract sap water from the xylem without significant isotopic fractionation, thus accurately reflecting the isotopic signal of transpiration water. CE might be the most promising alternative technique for extracting woody plant water for isotopic analysis because of its high extraction success rate. This study also confirmed that no isotopic fractionation occurred during root water uptake in Malus pumila Mill trees.