Cryogenic vacuum distillation-induced deuterium isotope biases in leaf water and their ecophysiological implications

Recent experiments show that cryogenic vacuum distillation (CVD) - the standard method for plant water extraction - can introduce biases into δ²H measurements of stem water. However, whether similar biases are present in leaf water remains unknown. To test for CVD biases in leaf water, we used an immersion-based rehydration approach to establish reference water isotope signatures in leaf and stem samples collected from 45 diverse field-grown species. By comparing isotopic ratios of reference and CVD-extracted waters, we demonstrate that CVD systematically underestimates leaf water δ²H in all species, with magnitudes comparable to those observed in stems. Moreover, the δ²H offsets for leaves and stems showed comparable negative correlations with tissue relative water content. By contrast, no CVD-caused offsets were observed in the control samples (quartz sand and cellulose triacetate) lacking exchangeable organic hydrogen (H). Our study provides the first evidence for the pervasive presence of CVD artifacts in leaf water and identifies deuterium exchange between organic matter and water as the main underlying mechanism. As discussed further, the observed artifacts could have important implications for interpreting fractionation mechanisms that shape δ²H values of leaf water and plant organic biomarkers - and thus for isotope-based ecological and paleoclimatic applications broadly.