| Abstract: | The hydrogen isotope ratio of water cryogenically extracted from plant stem samples (δ2Hstem_CVD) is routinely used to aid isotope applications that span hydrological, ecological, and paleoclimatological research. However, an increasing number of studies have shown that a key assumption of these applications—that δ2Hstem_CVD is equal to the δ2H of plant source water (δ2Hsource)—is not necessarily met in plants from various habitats. To examine this assumption, we purposedly designed an experimental system to allow independent measurements of δ2Hstem_CVD, δ2Hsource, and δ2H of water transported in xylem conduits (δ2Hxylem) under controlled conditions. Our measurements performed on nine woody plant species from diverse habitats revealed a consistent and significant depletion in δ2Hstem_CVD compared with both δ2Hsource and δ2Hxylem. Meanwhile, no significant discrepancy was observed between δ2Hsource and δ2Hxylem in any of the plants investigated. These results cast significant doubt on the long-standing view that deuterium fractionation occurs during root water uptake and, alternatively, suggest that measurement bias inherent in the cryogenic extraction method is the root cause of δ2Hstem_CVD depletion. We used a rehydration experiment to show that the stem water cryogenic extraction error could originate from a dynamic exchange between organically bound deuterium and liquid water during water extraction. In light of our finding, we suggest caution when partitioning plant water sources and reconstructing past climates using hydrogen isotopes, and carefully propose that the paradigm-shifting phenomenon of ecohydrological separation (“two water worlds”) is underpinned by an extraction artifact.The analysis of the stable isotope ratios of plant source water (δsource) is a powerful tool enabling the elucidation of a range of plant physiological, ecological, and hydrological processes from scales ranging from individual plants to the planet. δsource provides a foundation on which to form isotope signals of transpired water vapor and plant-derived biomarkers (i.e., cellulose and lipids) and thus is of high relevance to studies of terrestrial water fluxes (1, 2) and paleoclimate reconstructions (3, 4). δsource also contains information on the spatial and temporal origins of water used by plants and so is commonly used for investigating plant water uptake patterns under natural conditions (5, 6). Moreover, dual-isotope (δ2H and δ18O) analysis of δsource was critical in formulating the paradigm-shifting “two water worlds” (TWW) hypothesis, whereby ecohydrological separation exists between plant-accessible soil water pools and those recharging streams and groundwater (7, 8).Elucidation of the foregoing processes rest on the assumption that water extracted from plant stems is isotopically identical to water taken up by plant roots. Plant stem water is typically extracted with the cryogenic-vacuum distillation technique; δ generated with this method is hereinafter referred as δstem_CVD (9). For δstem_CVD to be an accurate indicator of δsource (i.e., δstem_CVD = δsource), two prerequisites must be met: 1) isotope change does not occur during root uptake and/or xylem transport of the source water (prerequisite I) and 2) stem water cryogenic extraction is a robust approach toward isotope recovery of xylem water (prerequisite II). The “δstem_CVD = δsource” assumption is generally valid for oxygen isotopes of water, but numerous studies have used hydrogen isotopes to assess source water, and here this assumption has faced scrutiny, as multiple studies have reported significant depletion in δ2Hstem_CVD compared with δ2Hsource in plants from various habitats (10–18).A frequently invoked explanation for the observed δ2Hstem_CVD depletion is a violation of prerequisite I, as it is believed that symplastic uptake of source water into the root xylem can give rise to hydrogen isotope fractionation (10, 11, 13, 19). The available evidence (10, 11) in support of such an explanation is largely peripheral, because direct, unambiguous confirmation of water uptake/transport-related fractionation would require a comparison of deuterium in source water and water transported within xylem conduits (δ2Hxylem). However, this type of comparison is difficult owing to the technical challenges in obtaining targeted measurements of δ2Hxylem in most plants. Intriguingly, in a field-grown riparian tree species (Populus euphratica) in which δ2Hxylem measurement was made possible with the aid of a syringe-aided xylem sap bleeding technique, no significant difference was observed between δ2Hxylem and δ2Hsource (12). This led to the suggestion that, at least for the investigated species, δ2Hstem_CVD depletion arises not from a violation of prerequisite I, but rather from a violation of prerequisite II. The violation of prerequisite II has been deemed possible (12, 17) based on the argument that hydrogen isotope heterogeneity could be present within the bulk stem water (i.e., the outside xylem water may carry a metabolism-induced, more-depleted δ2H signature compared with the xylem water), potentially causing the stem water extraction technique to artifactually underestimate δ2Hxylem.Given the controvertible state of knowledge regarding the mechanism driving δ2Hstem_CVD depletion, it is imperative for us to build a better and more comprehensive understanding of the isotopic relationships among cryogenic extracted bulk stem water, source water, and xylem water in different plants, so as to put the application of the stem water cryogenic extraction technique in diverse fields on firmer ground. In this context, it should be pointed out that the xylem water direct sampling technique (12) is applicable only to a few riparian tree species. Recently, a new method relying on laser-enabled isotope measurement of water vapor in equilibrium with xylem water has demonstrated potential for in situ continuous monitoring of xylem isotope signatures in trees (20, 21); however, the method needs further development before it becomes broadly applicable to different plant types. Thus, a more generally applicable method is needed for determining xylem water signature across diverse plant types.Toward this goal, and capitalizing on the well-recognized mass balance-dictated principle that the isotopic composition of steady-state (SS) plant transpiration is identical to that of the xylem water supplying the plant canopy, we custom-designed a measurement system to enable independent quantification of xylem water isotope composition through isotope measurement of SS plant transpiration. This measurement system conferred the ability to compare values of δstem_CVD, δsource, and δxylem across a number of plant species of varying native habitats. The data allowed us to confirm the common presence of δ2Hstem_CVD depletion across all plant types measured, and also to demonstrate that this phenomenon is caused by cryogenic extraction-associated artifact and not by water uptake/transport-related fractionation. We also performed a rehydration experiment to illustrate that the extraction artifact is unrelated to within-stem isotope heterogeneity as has been recently suggested, but rather is more likely linked to a deuterium-exchange process that occurs dynamically during cryogenic extraction. Using the TWW hypothesis as an example, we further discuss the ramifications for ecological/hydrological queries that rely on accurate isotopic information on plant source/xylem water. |
