Embolism propagation does not rely on pressure only: time-based shifts in xylem vulnerability curves of angiosperms determine the accuracy of the flow-centrifuge method

Centrifuges provide a fast approach to quantify the embolism resistance of xylem in vulnerability curves (VCs). Since embolism formation is assumingly driven by pressure only, spintime is not standardized for flowcentrifuge experiments. Here, we explore to what extent embolism resistance could be spin-time dependent and hypothesize that changes in hydraulic conductivity (Kh) would shift VCs towards higher water potential (Ψ) values over time. We quantified time-based shifts in flow-centrifuge VCs and their parameter estimations for six angiosperm species by measuring Kh over 15 min of spinning at a particular speed before a higher speed was applied to the same sample. We compared various VCs per sample based on cumulative spintime and modelled the relationship between Kh, Ψ and spin-time. Time-based changes of Kh showed considerable increases and decreases at low and high centrifuge speeds, respectively, which generally shifted VCs towards more positive Ψ values. Values corresponding to 50% loss of hydraulic conductivity (P50) became less negative by up to 0.72 MPa in Acer pseudoplatanus L., and on average by 8.5% for all six species compared with VCs that did not consider spin-time. By employing an asymptotic exponential model, we estimated time-stable Kh, which improved the statistical significance of VCs in five of the six species studied. This model also revealed the instability of VCs at short spin times with embolism formation in flowcentrifuges following a saturating exponential growth curve. Although pressure remains the major determinant of embolism formation, spin-time should be considered in flow-centrifuge VCs because not considering the time-dependent stability of Kh overestimates embolism resistance. This spin-time artefact is species-specific and likely based on relatively slow gas diffusion that is associated with embolism propagation. The accuracy of VCs is improved by determining time-stable Kh values for each centrifuge speed without considerably extending the experimental time to construct VCs.