Influence of sample preparation procedures on water stable isotopes in plant organs using the water-vapour equilibrium method

Water is a limiting factor for plant development. Therefore, understanding plant-water relations is vital for food security and ecosystem conservation. The stable isotopes of water (delta H-2 and delta O-18) are widely used to study ecohydrological processes, such as root water uptake. However, obtaining water from plants to measure their stable isotopic composition requires water extraction techniques that are laborious and challenging. A method developed for soil pore water that circumvents water extraction is the water-vapour equilibrium (WVE) method. In this study, we tested the capability and limits of WVE to determine stable water isotopes in different woody and non-woody plant organs. For this purpose, we analysed roots, stems, twigs, leaves and fruits of various plants. We first tested the effect of various equilibration times (24-72 h) and preparation techniques (cutting or grinding samples) on the measured isotope ratios. Analyser-internal variables that react to volatile organic compounds (VOCs) interfering with the measurement were also considered. Cutting samples and equilibrating for max. 24 h resulted in the most plausible isotope ratios, which was further tested as a proof-of-concept using controlled irrigation experiments. We could show expected progressive isotope enrichment from roots to leaves. Further, the isotopic composition of the water in fruit cores was more similar to irrigation water than that in fruit skins, and all the obtained results were consistent with the current process understanding of stable isotopes of water in plants, showing the feasibility of the chosen sample preparation. WVE seems to be a promising method to measure plant water isotopes with the challenge of dealing with VOCs likely influencing results.