Short-time xylem tension relaxation prevents vessel refilling and alleviates cryo-fixation artifacts in diffuse-porous Carpinus tschonoskii and Cercidiphyllum japonicum

Xylem tension relaxation is an important procedure that closely resembles the in vivo xylem water distribution when measuring conductivity or observing water distribution of plant tissue samples by cryo-scanning electron microscopy (cryo-SEM). Recent studies have shown that partial xylem embolism occurs when samples under tension are cut under water and that gas-filled vessels are refilled during tension relaxation. Furthermore, the frequency of gas-filled vessels has been reported to increase in samples without tension relaxation before cryo-fixation by liquid nitrogen, particularly in samples with significant tension. Here, we examined the effect of tension relaxation on these artifacts in Carpinus tschonoskii and Cercidiphyllum japonicum using magnetic resonance imaging. We observed that xylem embolism rarely occurs in bench-dried samples cut under water. In both species, a small portion of the xylem was refilled within ~1 h after tension relaxation. Cryo-SEM observations revealed that short-time (<1 h) xylem tension relaxation decreases the frequency of gas-filled vessels in samples frozen after xylem tension relaxation regardless of the water potential compared with that in samples frozen without rehydration in both species. Therefore, short-time tension relaxation is necessary to retain xylem water distribution during sample preparation against artifacts.