Waterlogging induces oxidative damage by accumulation of reactive oxygen species due to stomatal closure. Plants alter their physiological and molecular mechanisms to reduce and adapt to oxidative stress. This mechanism of adaptation to stress, known as hardening, can support future stress tolerance. Pinus densiflora seedlings were grown under waterlogging treatment for three years and then transplanted to another site to identify the waterlogging hardening effect on transplanting. Transcriptome analysis was conducted before and after transplanting, and physiological factors were measured after transplanting. After transplanting, wounding stress is the main cause of transplant stress, and 13 genes related to phenylpropanoid were upregulated for the recovery of wounded roots in waterlogged hardened seedlings. The leaf starch and soluble sugar content of the waterlogged hardened seedlings were 50.3% and 40.5% lower due to the formation of cell walls. However, auxin-related genes were downregulated in waterlogging hardened seedlings, resulting in a lower tendency for height growth in hardened waterlogged seedlings. Waterlogging hardening mitigated transplant stress by wounding more than non-hardening, whereas waterlogging hardening may negatively affect seedling height. Our study provides evidence for the hardening effect of long-term waterlogging on transplanted P. densiflora seedlings.

Stable isotopes of water (δ²H and δ¹⁸O) are reliable tracers for the investigation of plant–soil–water interactions in forest ecosystems. However, variations in isotopic compositions may arise due to differences in analytical instruments and water extraction methods. In this study, we conducted three different experiments to identify isotopic differences caused by analytical and methodological variations. First, we analyzed soil water by using the two most commonly applied methods: isotope ratio mass spectrometry (IRMS) and cavity ring-down spectroscopy (CRDS). Second, we compared the isotopes in xylem water extracted from the stems of nine tree species using cryogenic vacuum distillation (CVD) with different heating times. Third, we compared the compositions in xylem water extracted with three different methods: mechanical squeezing using a pressure chamber (PC), an induction module (IM), and CVD. The differences in isotopic composition between IRMS and CRDS were significant but minimal. Soil properties were not significant factors contributing to differences between the two instruments. For the xylem water extraction with CVD, each of the nine tree species required heating for more than three hours. Significant differences were observed in δ²H among the three extraction methods for xylem water. Xylem water extracted by CVD showed more depleted values compared to those obtained by PC and IM. Our results highlight the importance of considering analytical and methodological variations in stable isotope analysis.