Ageing-induced shrinkage of intervessel pit membranes in xylem of Clematis vitalba modifies its mechanical properties as revealed by atomic force microscopy

Abstract

Bordered pit membranes of angiosperm xylem are anisotropic, mesoporous media between neighbouring conduits, with a key role in long distance water transport. Yet, their mechanical properties are poorly understood. Here, we aim to quantify the stiffness of intervessel pit membranes over various growing seasons. By applying an AFM-based indentation technique “Quantitative Imaging” we measured the effective elastic modulus (Eeffective) of intervessel pit membranes of Clematis vitalba in dependence of size, age, and hydration state. The indentation-deformation behaviour was analysed with a non-linear membrane model, and paired with magnetic resonance imaging to visualise sap-filled and embolised vessels, while geometrical data of bordered pits were obtained using electron microscopy. Eeffective was transformed to the geometrically independent apparent elastic modulus Eapparent and to aspiration pressure Pb. The material stiffness (Eapparent) of fresh pit membranes was with 57 MPa considerably lower than previously suggested. The estimated pressure for pit membrane aspiration was 2.20+28 MPa. Pit membranes from older growth rings were shrunken, had a higher material stiffness and a lower aspiration pressure than current year ones, suggesting an irreversible, mechanical ageing process. This study provides an experimental-stiffness analysis of hydrated intervessel pit membranes in their native state. The estimated aspiration pressure suggests that membranes are not deflected under normal field conditions. Although absolute values should be interpreted carefully, our data suggest that pit membrane shrinkage implies increasing material stiffness, and highlight the dynamic changes of pit membrane mechanics and their complex, functional behaviour for fluid transport.

Angiosperms follow a convex trade‐off to optimize hydraulic safety and efficiency

“…We propose that water potential homeostasis, which in the short term is mainly controlled by stomatal and osmotic adjustments, can also be determined to some extent by interconduit pit characteristics, which are determined during the time of cambial division and conduit differentiation. Interestingly, ageing‐related changes in the pit membrane thickness within or across growing seasons will also affect the pit membrane porosity and its mechanical properties (Zhang et al ., 2020; Carmesin et al ., 2023), with consequences for flow across these mesoporous media.…”

“…6b) also presented slightly lower correlations with Ψ 50 than with pit membrane thickness. This may be explained by error propagation, since pit membrane thickness is the only trait directly measured, despite some imprecision caused by a low number of sample sizes (Kaack et al ., 2019), and possibly ageing‐induced shrinkage (Schmid & Machado, 1968; Sorek et al ., 2021; Carmesin et al ., 2023). On the contrary, the total pit membrane pit area cannot be directly measured yet, and depends on various anatomical estimations (Eqn 10), which means that a larger error propagation would be expected.…”

“…Interestingly, ageing-related changes in the pit membrane thickness within or across growing seasons will also affect the pit membrane porosity and its mechanical properties (Zhang et al, 2020;Carmesin et al, 2023), with consequences for flow across these mesoporous media.…”

“…Importantly, the reason why the intervessel pit area would be related to embolism vulnerability is that the likelihood of a leaky pit would increase with the total pit area (Pit Area Hypothesis; Wheeler et al, 2005;Hacke et al, 2006). However, there is no evidence of rare, leaky intervessel pits with large pores in hydrated pit membranes (Pesacreta et al, 2005;Carmesin et al, 2023), while a dehydration artefact appears to explain previous reports of large pores in dried pit membranes (Zhang et al, 2017). In addition, it is essential to consider the three-dimensional structure of pit membranes (Zhang et al, 2020), as well as the dynamic, potentially low surface tension due to the presence of polar lipids at gas-liquid interfaces of xylem sap (Schenk et al, 2021), which challenges earlier interpretations of embolism formation via a rare, large pore (Kaack et al, 2021).…”

Gold perfusion experiments support the multi‐layered, mesoporous nature of intervessel pit membranes in angiosperm xylem

“…The injection pressure was achieved with a Scholander pressure chamber (Model 1000 Pressure Chamber Instrument; PMS Instrument Co., Albany, OR, USA). Also, the 6 and 200 kPa injection pressures were unlikely to cause mechanical compression, aspiration, or deformation of pit membranes (Tixier et al ., 2014; Carmesin et al ., 2023).…”