We assessed how the seasonal variability of precipitation δ H and δ O is propagated into soil and xylem waters of temperate trees, applied a hydrological model to estimate the residence time distribution of precipitation in the soil, and identified the temporal origin of water taken up by Picea abies and Fagus sylvatica over 4 yr. Residence times of precipitation in the soil varied between a few days and several months and increased with soil depth. On average, 50% of water consumed by trees throughout a year had precipitated during the growing season, while 40% had precipitated in the preceding winter or even earlier. Importantly, we detected subtle differences with respect to the temporal origin of water used by the two species. We conclude that both current precipitation and winter precipitation are important for the water supply of temperate trees and that winter precipitation could buffer negative impacts of spring or summer droughts. Our study additionally provides the means to obtain realistic estimates of source water δ H and δ O values for trees from precipitation isotope data, which is essential for improving model-based interpretations of δ O and δ H values in plants.

Temperate tree species differ in their physiological sensitivity to declining soil moisture and drought. Although species-specific responses to drought have often been suggested to be the result of different water uptake depths, empirical evidence for such a mechanism is scarce. Here we test if differences in water uptake depths can explain previously observed species-specific physiological responses of temperate trees to drought and if the water uptake depth of different species varies in response to declining soil moisture. For this purpose, we employed stable oxygen and hydrogen isotopes of soil and xylem water that we collected over the course of three growing seasons in a mature temperate forest in Switzerland. Our data show that all investigated species utilise water from shallow soil layers during times of sufficient soil water supply. However, Fraxinus excelsior, Fagus sylvatica and Acer pseudoplatanus were able to shift their water uptake to deeper soil layers when soil water availability decreased in the topsoil. In contrast, Picea abies, was not able to shift its water uptake to deeper soil layers. We conclude from our data that more drought-resistant tree species are able to shift their water uptake to deeper soil layers when water availability in the topsoil is becoming scarce. In addition, we were able to show that water uptake depth of temperate tree species is a trait with high plasticity that needs to be characterised across a range of environmental conditions.

Temperate forests are expected to be particularly vulnerable to drought and soil drying because they are not adapted to such conditions and perform best in mesic environments. Here we ask (i) how sensitively four common temperate tree species (Fagus sylvatica, Picea abies, Acer pseudoplatanus and Fraxinus excelsior) respond in their water relations to summer soil drying and seek to determine (ii) if species-specific responses to summer soil drying are related to the onset of declining water status across the four species. Throughout 2012 and 2013 we determined tree water deficit (TWD) as a proxy for tree water status from recorded stem radius changes and monitored sap flow rates with sensors on 16 mature trees studied in the field at Lägeren, Switzerland. All tree species responded equally in their relative maximum TWD to the onset of declining soil moisture. This implies that the water supply of all tree species was affected by declining soil moisture and that none of the four species was able to fully maintain its water status, e.g., by access to alternative water sources in the soil. In contrast we found strong and highly species-specific responses of sap flow to declining soil moisture with the strongest decline in P. abies (92%), followed by F. sylvatica (53%) and A. pseudoplatanus (48%). F. excelsior did not significantly reduce sap flow. We hypothesize the species-specific responses in sap flow to declining soil moisture that occur despite a simultaneous increase in relative TWD in all species reflect how fast these species approach critical levels of their water status, which is most likely influenced by species-specific traits determining the hydraulic properties of the species tree.

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.

<p>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 (<em>δ</em><sup>18</sup>O) is therefore essential.</p><p>We aimed to resolve the impact of different steps on sugars' <em>δ</em><sup>18</sup>O 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 <sup>18</sup>O-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 (<em>δ</em><sup>13</sup>C).</p><p>Comparison of freeze- and oven-dried sugars showed lower <em>δ</em><sup>18</sup>O 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, <sup>18</sup>O-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' <em>δ</em><sup>13</sup>C values.</p><p>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 <em>δ</em><sup>18</sup>O 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.</p>

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